Peptides and compositions for treatment of joint damage

ABSTRACT

The present invention provides new protease-resistant polypeptides, as well as compositions and methods for treating, ameliorating or preventing conditions related to joint damage, including acute joint injury and arthritis.

BACKGROUND OF THE INVENTION

Osteoarthritis (OA) represents the most common musculoskeletal disorder.Approximately 40 million Americans are currently affected; a numberpredicted to increase to 60 million within the next twenty years as aresult of aging population and an increase in life expectancy, making itthe fourth leading cause of disability. OA is characterized by a slowdegenerative breakdown of a joint including both articular cartilage(containing the cells and matrix which produce lubrication andcushioning for the joint) and subchondral bone underlying the articularcartilage. OA can be considered a consequence of various etiologicfactors. For example, it can be caused by abnormal biomechanical stressor genetic or acquired abnormalities of articular cartilage or bone.Current OA therapies include pain relief with oral NSAIDs or selectivecyclooxygenase 2 (COX-2) inhibitors, intra-articular (IA) injection withagents such as corticosteroids and hyaluronan, and surgical approaches.

Joint damage, e.g., acute joint injury, such as a meniscal or ligamenttear, or an intra-articular fracture can also lead to arthritis, e.g.,posttraumatic arthritis. Because articular cartilage has a limitedability to repair, even small undetectable damage can often get worseover time and lead to OA. Current treatments for joint injury caninclude surgery and other invasive procedures focused on regeneration ofdamaged joints as well as treatment with agents to reduce pain andinflammation.

Mesenchymal stem cells (MSCs) are present in adult articular cartilageand upon isolation can be programmed in vitro to undergo differentiationto chondrocytes and other mesenchymal cell lineages, and may be used forcartilage regeneration. In part, the process is regulated by growthfactors (TGFβs, BMPs), serum conditions and cell-cell contact.WO2011/008773 describes peptide compositions and use of thosecompositions for treating or preventing arthritis and joint injury andfor inducing differentiation of mesenchymal cells into chondrocytes.Additionally, WO2012/129562 describes small molecule compounds,compositions and use of those compositions for amelioration of arthritisand joint injury and for inducing differentiation of mesenchymal cellsinto chondrocytes.

Though surgical techniques, and regenerative technology have made someprogress in restoration of cartilage, slowing degeneration, and improvedrepair of joint damage, a continued need exists for improvement ofcompositions and methods for effective cartilage regeneration, treatmentof joint damage and amelioration or prevention of OA.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to the identification of new polypeptideand protein variants of angiopoietin-like 3 (ANGPTL3) that have improvedpharmaceutical properties, e.g., are more stable, less susceptible toproteolysis and enzymatic degradation than wild-type ANGPTL3. Alsoprovided are pharmaceutical compositions and methods for treatment ofjoint damage or joint injury, and methods of ameliorating or preventingarthritis, joint damage or joint injury in a mammal.

Thus, provided are protease-resistant polypeptides comprising an aminoacid sequence that has at least 95% amino acid sequence identity, or atleast 96%, 97%, 98%, 99% or 100% amino acid sequence identity to anamino acid sequence selected from any one or more of the sequences ofTABLE 1, and as further described herein. The modified polypeptides ofTABLE 1 include an amino acid that is a polar amino acid other than K orR at position 423, as determined with reference to the full lengthANGPTL3 polypeptide sequence, SEQ ID NO:1. In some embodiments the aminoacid at position 423 as determined with reference to SEQ ID NO:1 is Q orS. In certain embodiments the amino acid at position 423 as determinedwith reference to SEQ ID NO:1 is Q. In certain embodiments the aminoacid at position 423 as determined with reference to SEQ ID NO:1 is S.In certain embodiments the amino acid at position 423 as determined withreference to SEQ ID NO:1 is deleted. In addition, provided polypeptideshave chondrogenic activity.

In some embodiments, the polypeptide comprises a sequence having atleast 95% identity or at least 96%, 97%, 98%, 99% or 100% to any one ofSEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70. In some embodiments thepolypeptide comprises a sequence having at least 95% identity or atleast 96%, 97%, 98%, 99% or 100% to any one of SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:63, and SEQ ID NO:64. In some embodiments, the polypeptide comprisesany one of the sequences of TABLE 1. In some embodiments, thepolypeptide comprises any one of SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQID NO:70. In some embodiments the polypeptide comprises any one of SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:58, SEQ IDNO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, and SEQID NO:64. In some embodiments, the polypeptide is any one of thesequences of TABLE 1. In some embodiments, the polypeptide is any one ofSEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70. In some embodiments thepolypeptide is any one of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ IDNO:62, SEQ ID NO:63, and SEQ ID NO:64.

Polypeptides of the invention may incorporate one or more chemicalmodifications (e.g., PEGylation). In some embodiments, polypeptides ofthe invention may comprise a heterologous peptide as a fusion protein,which may optionally be fused at the amino-terminal or thecarboxy-terminal end of the polypeptide. Also provided arepolynucleotides encoding the polypeptides of the invention; vectorscontaining polynucleotides encoding the polypeptides; and host cellscomprising such vectors.

The present invention also provides pharmaceutical compositionscomprising the polypeptides of the invention and a pharmaceuticallyacceptable carrier. Such compositions can be used in methods providedherein for treating, ameliorating or preventing arthritis or jointdamage in a patient, where the method comprises administering to a jointof a patient a therapeutically effective amount of a pharmaceuticalcomposition of the invention. Examples of conditions that can benefitfrom such methods include, but are not limited to arthritis (e.g.,osteoarthritis, traumatic arthritis), and joint damage (e.g., acutejoint injury).

The present invention further provides methods of treating a subjectcomprising administering a therapeutically effectively amount of apolypeptide of the invention. Provided methods include treating asubject having or at risk of having joint damage and/or arthritis,comprising administering to the subject a therapeutically effectiveamount of one or more polypeptides of the invention or a pharmaceuticalcomposition thereof. Still further provided are methods of inducingdifferentiation of mesenchymal stem cells into chondrocytes, comprisingcontacting mesenchymal stem cells with an effective amount of apolypeptide of the invention to induce differentiation of themesenchymal stem cells into chondrocytes.

These and other aspects of the invention, including additional features,advantages, and embodiments of the invention, will be described andelucidated in further detail in the following detailed description andappended claims of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic of hANGPTL3 proteins engineered to improveprotein stability and enhance proteolytic resistance. During proteinproduction of wild type protein and peptide sequences, 100% cleavage wasobserved between Lys423 and Ser424. To mitigate proteolysis, variousmutant peptides were generated wherein Lys 423 was mutated to Gln orSer; or Ser424 was mutated to Thr; or Lys 423 was deleted.

FIGS. 2A and B depicts graphical representations of expression ofcartilage specific proteins in the presence or absence of ANGPTL3 andengineered constructs. Fixed cells were stained for 2A Pro-collagen Type2A quantification (PIIANP) or 2B Type II collagen quantification todetermine the % of cells differentiating into chondrocytes followingtreatment as described in the Exemplification. FIG. 2C depicts graphicalrepresentation of quantification of angiogenesis assays in the presenceor absence of ANGPTL3 or engineered construct as compared to a positivecontrol protein, bFGF. Total tube length and number of branch pointswere quatitative measurements of angiogenesis. Although others havereported angiogenic activity in ANGPTL3, and this study confirmsactivity as well as that of FGF; the results indicated no significantactivity is retained in a Cterminal ANGPTL3 construct.

FIG. 3 are graphical representations showing an increase in expressionof cartilage specific proteins in the presence of ANGPTL3 or engineeredconstructs. 3A. Cells were evaluated ten days following treatment usingqRT-PCR to measure RNA expression for cartilage specific proteinsfollowing treatment as described. Lubricin, aggrecan and Sox9 representcartilage related proteins; IGF and IFITM1 represent differentiationpotential, and osteocalcin and type X collagen represent bone/fibrosisrelated proteins. 3B. Cells were evaluated three days followingtreatment as described. Increased aggrecan expression was seen followingtreatment with engineered construct or wild type ANGPTL1 C-terminalregion polypeptide.

FIG. 4 depicts graphical representations of chondro-protective activityof ANGPTL3 and engineered constructs. 4A: Glycosaminoglycan (GAG)release, an indicator of matrix damage, was inhibited with increasingamount of ANGPTL3 and mutant constructs. Ex vivo GAG release (anindicator of matrix damage) inhibition assays were performed usingbovine cartilage treated in the presence or absence of constructs asdescribed. 4B and 4C: NO release was inhibited with increasing amount ofANGPTL3 and engineered constructs as indicated. Chondrocytes weretreated in the presence or absence of constructs as described followedby Greiss reaction assays to determine the inhibition of NO release asan indicator of chondroprotection.

FIG. 5 depicts a graphical representation showing an inhibition of typeX collagen expression (an indicator of fibrotic cartilage formationactivity) in the presence of constructs under hypertrophic conditions.Primary chondrocytes were treated in the presence of absence ofconstructs under hypertrophic conditions as described, followed bydetermination of type X collagen expression, assessed byimmunofluorescence, as a measurement of formation of fibrotic andhypertrophic cartilage/chondrocyte differentiation. 5A depicts resultsof wild type C-terminal ANGPTL3 or engineered construct. 5B depictsresults of C-terminal ANGPTL3 (WT) or engineered constructs 242KQ or242Kde1 or C-terminal ANGPTL1.

FIG. 6 depicts a schematic representation of the dosing paradigm (6A),followed by a graphical representation (6B) of the improvement in jointseverity after treatment with mouse ANGPTL3 (17-460) as measured bycartilage erosion score of the lateral femoral condyle.

FIG. 7. is a graphical representation of incapacitance measurements (anindicator of pain) in mice following surgical induction of cartilagedamage and subsequent treatment with ANGPTL3 constructs once weekly forthree weeks (beginning on day7). 7A represents incapacitancemeasurements on day 35 following surgery; and 7B represents measurementstaken on day 56 following surgery.

FIG. 8. is a graphical representation of the total joint severity scoreand improvement in severity to cartilage damage induced by collagenasein mice following 3 once weekly treatments (days 7, 14 and 21) ofANGPTL3 constructs (indicated).

FIG. 9. depicts results in a rat meniscal tear model of joint damagefollowing treatment with engineered ANGPTL3 construct. FIG. 9A is agraphical representation of the proteoglycan content in joints fiveweeks following treatment; FIG. 9B is a graphical representation of thefemoral joint severity score five weeks following treatment. Resultsillustrate improvement to cartilage damage induced by surgical severingof the meniscus in rats following 3 once weekly treatments (days 7, 14and 21) of ANGPTL3 constructs (indicated).

FIG. 10 depicts results in a rat meniscal tear model of joint damagefollowing treatment with engineered ANGPTL3 construct. FIG. 10A is agraphical representation of percent of in vivo repair as measured byseverity, safranin 0 intensity, cartilage area and cartilage thickness.FIG. 10B is a graphical representation of of incapacitance measurements(an indicator of pain) in rats following surgical induction of cartilagedamage and subsequent treatment.

FIG. 11 is a graphical representation of the total gross severity scoreto illustrate improvement of cartilage damage induced by surgicaldisruption of the medial meniscus in dogs following biweekly dosingbeginning on day 4 (each of the 1.5 ug/dose or 15 ug/dose) or a single30 ug dose) given on day 7 only.

FIG. 12 provides a sequence alignment of the C-terminal domains of humanangiopoeitin like family membershANGPTL1(271-491)/hANGPTL3(241-460)/hANGPTL4(179-406)

DETAILED DESCRIPTION

The present invention is based, at least in part, on the identificationof Angiopoietin-like 3 (ANGPTL3) polypeptides that stimulate chondrocytedifferentiation of mesenchymal stem cells and that are resistant tocleavage by proteases (e.g., trypsin-like proteases). WO2011/008773,describes ANGPTL3 peptide compositions and use of peptide compositionsfor treating or preventing arthritis and joint injury and for inducingdifferentiation of mesenchymal cells into chondrocytes. We found thatwild type ANGPTL3 proteins are subject to protease clipping andinstability and have identified sequence variants to mitigate thiseffect. The present invention thus provides improved peptidecompositions for repairing cartilage. In particular, provided areANGPTL3 peptides modified in accordance with the present invention tohave increased protease-resistance as compared to a wild-type ANGPTL3polypeptide. Also provided are compositions and methods foradministration of ANGPTL3 polypeptides to prevent or amelioratearthritis or joint injury by administering a polypeptide of theinvention into a joint, a cartilage tissue or a cartilage proximaltissue, or systemically. Further, the invention provides compositionsand methods for induction of mesenchymal stem cell differentiation intochondrocytes.

Definitions

The term “protease-resistant” as used herein refers to a polypeptidecomprising a modification that renders the polypeptide less susceptibleto cleavage by a trypsin-like protease than a corresponding non-modifiedwildtype polypeptide. In specific embodiments a protease-resistantpolypeptide is an ANGPTL3 polypeptide that has an amino acidsubstitution, relative to a native wildtype peptide sequence, at an R ora K residue.

“ANGPTL3” refers to a member of the angoipoietin protein family. Anamino acid sequence of ANGPTL3 (GenBank Accession No. NP 055310.1) isset forth in SEQ ID NO:1; and the corresponding polynucleotide sequenceof which is set forth as SEQ ID NO: 2 (NCBI reference sequence numberNM014495.2, wherein the ANGPTL3 coding sequence comprises nt 52-1434 ofSEQ ID NO:2). “ANGPTL3 polypeptide” refers to a naturally occurringexpressed polypeptide. For the purposes of the present disclosure, thenumbering of an amino acid is typically determined with reference to thefull-length wildtype human ANGPTL3 polypeptide sequence (SEQ ID NO:1).Thus, in embodiments in which a polypeptide of the invention containsonly a C-terminal portion of full-length ANGPTL3, but not the N-terminalportion, although the peptide is less than 460 amino acids in length,the numbering of the positions is based on SEQ ID NO:1. For example,reference to position 423 of an ANGPTL3 polypeptide of the inventionrefers to position 423 of SEQ ID NO:1, even though the ANGPTL3polypeptide of the invention itself may only be 200 amino acids inlength. In determining an amino acid in a sequence of interest that“corresponds to” a position in a reference sequence, such as SEQ IDNO:1, this is performed by optimally aligning the sequences, e.g., usingthe default CLUSTAL alignment parameters or default BLAST 2 alignmentparameters and comparing the sequences. For example, position 423 in asequence of interest that is “determined with reference to SEQ ID NO:1”,or an amino acid that “corresponds to” position 423 of SEQ ID NO:1,means the amino acid that aligns with position 423 of SEQ ID NO:1 whenthe sequence of interest is optimally aligned with SEQ ID NO:1.

The terms “peptidomimetic” and “mimetic” refer to a synthetic chemicalcompound that has substantially the same functional characteristics of anaturally or non-naturally occurring polypeptide (e.g., ANGPTL3), butdifferent (though typically similar) structural characteristics. Peptideanalogs are commonly used in the field as non-peptide active compounds(e.g., drugs) with properties analogous to those of a template peptide.Such non-peptide compounds are termed “peptide mimetics” or“peptidomimetics” (Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber andFreidinger TINS p. 392 (1985); and Evans et al. J. Med. Chem. 30:1229(1987)). Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalent orenhanced therapeutic or prophylactic effect. Generally, peptidomimeticsare structurally similar to a paradigm polypeptide (i.e., a polypeptidethat has a biological or pharmacological activity), such as found in apolypeptide of interest, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of,e.g., —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans), —COCH₂—,—CH(OH)CH₂—, and —CH₂SO—. A mimetic can be either entirely composed ofsynthetic, non-natural analogues of amino acids, or, is a chimericmolecule of partly natural peptide amino acids and partly non-naturalanalogs of amino acids. A mimetic can also incorporate any amount ofnatural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. For example, a mimetic composition is within the scopeof the invention if it is capable of chondrogenic activity of an ANGPTL3polypeptide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. Polypeptides,peptides, and proteins of the invention comprise protease resistantANGPTL3 peptidomimetics having chondrogenic activity.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Naturally encoded amino acids arethe 20 common amino acids (alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine) as well as pyrrolysine,pyrroline-carboxy-lysine, and selenocysteine.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every polypeptidesequence herein which is encoded by a polynucleotide encompasses everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

One of skill will recognize that individual substitutions, deletions oradditions to a nucleic acid, peptide, polypeptide, or protein sequencewhich alters, adds or deletes a single amino acid or a small percentageof amino acids with reference to an original encoded amino acid sequenceresults in a “conservatively modified variant” where the alterationproduces substitution of an amino acid with a chemically similar aminoacid and/or a polypeptide sequence that produces a structurally similarprotein having similar functional activity to the original protein.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The term “conservative amino acid substitutions” refers to thesubstitution (conceptually or otherwise) of an amino acid from one suchgroup with a different amino acid from the same group. One example ofsubstitutions is based on analyzing the normalized frequencies of aminoacid changes between corresponding proteins of homologous organisms(see, e.g., Schulz, G. E. and R. H. Schirmer, Principles of ProteinStructure, Springer-Verlag). According to such analyses, groups of aminoacids may be defined where amino acids within a group exchangepreferentially with each other and, therefore, resemble each other mostin their impact on the overall protein structure (see, e.g., Schulz, G.E. and R. H. Schirmer, Principles of Protein Structure,Springer-Verlag). One example of a set of amino acid groups defined inthis manner include: (i) a charged group, consisting of Glu and Asp,Lys, Arg and His; (ii) a positively-charged group, consisting of Lys,Arg and His; (iii) a negatively-charged group, consisting of Glu andAsp; (iv) an aromatic group, consisting of Phe, Tyr and Trp; (v) anitrogen ring group, consisting of His and Trp; (vi) a large aliphaticnonpolar group, consisting of Val, Leu and Ile; (vii) a slightly-polargroup, consisting of Met and Cys; (viii) a small-residue group,consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro; (ix) analiphatic group consisting of Val, Leu, Ile, Met and Cys; and (x) asmall hydroxyl group consisting of Ser and Thr. Other examples ofconservative substitutions based on shared physical properties are thesubstitutions within the following groups: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the amino acid sequence or polynucleotide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (e.g., a polypeptide of theinvention), which does not comprise additions or deletions, for optimalalignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same sequences. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 95%identity, optionally 96%, 97%, 98%, or 99% identity over a specifiedregion, or, when not specified, over the entire sequence), when comparedand aligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Theinvention provides polypeptides that are substantially identical to thepolypeptides, respectively, exemplified herein (e.g., any of SEQ ID NOs:11-42), as well as uses thereof including but not limited to use fortreating or preventing arthritis or joint injury. Optionally, fornucleic acids, the identity exists over a region that is at least about150 nucleotides in length, or more preferably over a region that is 300to 450 or 600 or more nucleotides in length, or the entire length of thereference sequence. For amino acid sequence, optionally, identity existsover a region that is at least about 50 amino acids in length, or morepreferably over a region that is 100 to 150 or 200 or more amino acidsin length, or the entire length of the reference sequence.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 50 to 600, usually about 75 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443,by the search for similarity method of Pearson and Lipman (1988) Proc.Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA), or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is purified to be essentially free ofother cellular components with which it is associated in the naturalstate. It is often in a homogeneous or nearly homogeneous state. It canbe in either a dry or aqueous solution. Purity and homogeneity may bedetermined using analytical chemistry techniques known and usedtypically in the art, e.g., polyacrylamide gel electrophoresis, highperformance liquid chromatography, etc. A protein that is thepredominant species present in a preparation is substantially purified.The term “purified” in some embodiments denotes that a protein givesrise to essentially one band in an electrophoretic gel. Typically, itmeans that a protein is at least 85% pure, more preferably at least 95%pure, and most preferably at least 99% pure.

The term “hyaluronic acid” are used herein to include derivatives ofhyaluronic acid that include esters of hyaluronic acid, salts ofhyaluronic acid and also includes the term hyaluronan. The designationalso includes both low and high molecular weight forms of hyaluronansand crosslinked hyaluronans or hylans. Examples of such hyaluronans areSynvisc™ (Genzyme Corp. Cambridge, Mass.), ORTHOVISC™ (AnikaTherapeutics, Woburn, Mass.), HYALGAN™ (Sanofi-Synthelabo Inc., Malvern,Pa.), and ProVisc (Alcon/Novartis).

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contextclearly dictates otherwise.

Angiopoietin-Like 3 Protease-Resistant Polypeptides

Angiopoietin-like 3 is a member of the angiopoietin-like family ofsecreted factors. It is predominantly expressed in the liver, and hasthe characteristic structure of angiopoietins, consisting of a signalpeptide, N-terminal coiled-coil domain (CCD) and the C-terminalfibrinogen (FBN)-like domain. Angiopoietin-like 3 was shown to bindαV/β3 integrins and FBN-like domain alone was sufficient to induceendothelial cell adhesion and in vivo angiogenesis (Camenisch et al., J.Biol. Chem. 277: 17281-17290, 2002). Endogenous ANGPTL3 is generallycleaved in vivo into amino-terminal and carboxy-terminal fragments. Assummarized above and further described herein, the present inventioncontemplates use of various protease-resistant ANGPTL3 proteins havingchondrogenic activity.

In some embodiments, an isolated polypeptide comprises an amino acidsequence that has at least 95% identity, or at least 96%, 97%, 98%, or99% identity, to an amino acid sequence selected from any one of thesequences of TABLE 1, wherein the polypeptide comprises an amino acidthat is a polar amino acid other than K or R at position 423 or thepolypeptide comprises a deletion at position 423, as determined withreference to SEQ ID NO:1. The polypeptides of the invention havechondrogenic activity. In some embodiments, a polypeptide comprises theamino acid sequence that has at least 95% identity, or at least or atleast 96%, 97%, 98%, or 99% identity, to an amino acid sequence selectedfrom any one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:65, SEQ ID NO:66,SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70. wherein thepolypeptide comprises an amino acid that is a polar amino acid otherthan K or R at position 423 or the polypeptide comprises a deletion atposition 423, as determined with reference to SEQ ID NO:1, and thepolypeptide has chondrogenic activity. In a further embodiment, apolypeptide comprises the amino acid sequence that has at least 95%identity, or at least or at least 96%, 97%, 98%, or 99% identity, to anamino acid sequence selected from any one of SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:58,SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, orSEQ ID NO:64 wherein the polypeptide comprises an amino acid that is apolar amino acid other than K or R at position 423, as determined withreference to SEQ ID NO:1, and the polypeptide has chondrogenic activity.

In some embodiments, an isolated polypeptide comprises an amino acidsequence selected from any one of the sequences of TABLE 1, wherein thepolypeptide comprises an amino acid that is a polar amino acid otherthan K or R at position 423 or the polypeptide comprises a deletion atposition 423, as determined with reference to SEQ ID NO:1, and thepolypeptide has chondrogenic activity. In some embodiments, apolypeptide comprises an amino acid sequence selected from any one ofSEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70 wherein the polypeptidecomprises an amino acid that is a polar amino acid other than K or R atposition 423 or the polypeptide comprises a deletion at position 423, asdetermined with reference to SEQ ID NO:1, and the polypeptide haschondrogenic activity. In a further embodiment, a polypeptide comprisesan amino acid sequence selected from any one of SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:63, or SEQ ID NO:64 wherein the polypeptide comprises an amino acidthat is a polar amino acid other than K or R at position 423, asdetermined with reference to SEQ ID NO:1, and the polypeptide haschondrogenic activity.

In some embodiments, an isolated polypeptide has at least 95% identity,or at least 96%, 97%, 98%, or 99% identity, to an amino acid sequenceselected from any one of the sequences of TABLE 1, wherein thepolypeptide comprises an amino acid that is a polar amino acid otherthan K or R at position 423 or the polypeptide comprises a deletion atposition 423, as determined with reference to SEQ ID NO:1, and thepolypeptide has chondrogenic activity. In some embodiments, apolypeptide has at least 95% identity, or at least or at least 96%, 97%,98%, or 99% identity, to an amino acid sequence selected from any one ofSEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70, wherein the polypeptidecomprises an amino acid that is a polar amino acid other than K or R atposition 423 or the polypeptide comprises a deletion at position 423, asdetermined with reference to SEQ ID NO:1, and the polypeptide haschondrogenic activity. In a further embodiment, a polypeptide has atleast 95% identity, or at least or at least 96%, 97%, 98%, or 99%identity, to an amino acid sequence selected from any one of SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ IDNO:62, SEQ ID NO:63, or SEQ ID NO:64 wherein the polypeptide comprisesan amino acid that is a polar amino acid other than K or R at position423, as determined with reference to SEQ ID NO:1, and the polypeptidehas chondrogenic activity.

In some embodiments, an isolated polypeptide is an amino acid sequenceselected from any one of the sequences of TABLE 1. In some embodiments,a polypeptide is an amino acid sequence selected from any one of SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, or SEQ ID NO:70. In a further embodiment, apolypeptide is an amino acid sequence selected from any one of SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:58, SEQ IDNO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, or SEQ IDNO:64.

TABLE 1 ANGPTL3 variant constructs SEQ ID Construct Sequence 14 207KQIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 15 207KSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 16 225KQTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTDSESFE17 225KS TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTDSESFE18 225ST TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKTKPERRRGLSWKSQNGRLYSI KSTKMLIHPTDSESFE19 226KQ TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIK STKMLIHPTDSESFE20 226KS TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIK STKMLIHPTDSESFE21 228KQ FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKST KMLIHPTDSESFE 22228KS FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKST KMLIHPTDSESFE 23228ST FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKTKPERRRGLSWKSQNGRLYSIKST KMLIHPTDSESFE 24233KQ EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTDSESFE 25233KS EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTDSESFE 26241KQ GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 27 241KSGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 28 242KQIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 29 242KSIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 30 225-455KQTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTD 31225-455KS TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTD 32226-455KQ TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIK STKMLIHPTD 33226-455KS TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIK STKMLIHPTD 34228-455KQ FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKST KMLIHPTD 35228-455KS FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKST KMLIHPTD 36233-455KQ EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTD 37 233-455KSEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTD 38 241-455KQGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 39 241-455KSGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 40 242-455KQIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 41 242-455KSIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 58 207KdelIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 59 225KdelTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIK STKMLIHPTDSESFE60 226Kdel TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKS TKMLIHPTDSESFE61 228Kdel FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTK MLIHPTDSESFE 62233Kdel EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHP TDSESFE 63241Kdel GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 64 242KdelIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 65 225-TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG 455KdelSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIK STKMLIHPTD 66226- TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS 455KdelPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKS TKMLIHPTD 67228- FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW 455KdelTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTK MLIHPTD 68 233-EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH 455KdelRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHP TD 69 241-GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF 455KdelNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 70 242-IPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN 455KdelETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD

Modified ANGPTL3 polypeptides of the invention have at least onesubstitution in the C-terminal portion of the polypeptide to render thepolypeptide protease resistant. The substitution is at an R or K residueso that polypeptides have increased resistance, e.g., to trypsin-likeproteases. Any amino acid may be substituted for an R or K in a proteaseresistant ANGPTL3 polypeptide of the invention. In some embodiments, asubstitution is a polar amino acid, e.g., H, N, Q, S, T, A, or Y. Insome embodiments, a substitution is H, N, Q, S, T, or Y. In someembodiments, a substitution is S or Q. In some embodiments, thesubstitution is Q. In some embodiments the substitution is S. In someembodiments, a protease-resistant peptide has an amino acid at position423, with reference to SEQ ID NO:1, that is other than K or R. In someembodiments, a polypeptide of the invention comprises an amino acid atposition 423 that is a polar amino acid. For example, the amino acid atposition 423 may be Q or S or another polar amino acid. In certainembodiments a polypeptide of the invention has a Q at position 423. Inother embodiments a polypeptide of the invention has an S at position423. In some embodiments, in addition to substitution at 423, theprotease-resistant peptide has a substitution of another R or K in theC-terminus of SEQ ID NO:1, or a variant thereof, wherein thesubstitution is a polar amino acid other than R or K. In someembodiments, the substitution at position 423 as determined withreference to SEQ ID NO:1, is Q or S. In still other embodiments apolypeptide of the invention has a deletion at position 423 asdetermined with reference to SEQ ID NO:1.

In some embodiments, a polypeptide of the invention is 250 amino acidsor less in length and comprises the amino acid sequence of SEQ ID NO:16,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63,SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68,SEQ ID NO:69, or SEQ ID NO:70.

In some embodiments, the invention provides for use of full-lengthprotease-resistant, chondrogenic ANGPTL3 proteins. In some embodiments,the invention provides for protease-resistant ANGPTL3 proteinscomprising a C-terminal portion of the ANGPTL3 sequence, or achondrogenic variant thereof. In certain embodiments ANGPTL3 proteinslack the amino-terminal end of the native protein. In some embodiments,protease-resistant ANGPTL3 proteins of the invention lack the CCD domainand/or lacks significant CCD activity. Thus, in some embodiments, theprotease-resistant ANGPTL3 proteins of the invention comprise at least afragment (e.g., at least 100, 150, 200, 220 or 215 contiguous aminoacids) of a human ANGPTL3 protein carboxy-terminal domain, or asubstantially identical sequence to the human carboxy-terminal ANGPTL3protein sequence, wherein the polypeptide and variants thereof retainschondrogenic activity. In some embodiments, a protease-resistantpolypeptide of the invention lacks at least a portion of the C-terminalsequence, e.g., lacks 5, 10, 15, or 20 amino acids from the C-terminalend of SEQ ID NO:1 (i.e., lacks 456-460, 451-460, 446-460 or 441-460 ofSEQ ID NO:1).

In some embodiments, a protease-resistant ANGPTL3 polypeptide of theinvention comprises contiguous amino acids corresponding to the aminoacid regions: amino acids 241-455, or 241-460 of SEQ ID NO:1; aminoacids 242-455, or 242-460 of SEQ ID NO:1; amino acids 233-455 or 233-460of SEQ ID NO:1; amino acids 228-455 or 228-460 of SEQ ID NO:1, aminoacids 226-455- or 226-260 or amino acids 225-455- or 225-260 of SEQ IDNO:1 in which an amino acid is substituted for an R or K or a singleresidue is deleted. In some embodiments, a substitution is at position423 as determined with reference to SEQ ID NO:1. In some embodiments adeletion is at position 423 as determined with reference to SEQ ID NO:1.In some embodiments, a protease-resistant polypeptide comprisescontiguous amino acids corresponding to the amino acid regions 207-455or 207-460 of SEQ ID NO:1 in which an amino acid is substituted for R orK or a single residue is deleted. In some embodiments, a substitution ordeletion is at position 423. In some embodiments, a substitution is apolar amino acid, e.g., H, N, Q, S, T, A, or Y. In some embodiments, asubstitution is H, N, Q, S, T, or Y. In some embodiments, a substitutionis S or Q. In some embodiments, a substitution is Q. In certainembodiments a deletion at position 423 relative to SEQ ID NO:1 isincluded.

The invention additionally provides a protease-resistant polypeptide,wherein the polypeptide comprises an amino acid sequence having at least95% identity, or at least 96%, 97%, 98%, or 99% identity, to amino acids240-454 of SEQ ID NO:1, amino acids 241-455 of SEQ ID NO:1, or aminoacids 242-455 of SEQ ID NO:1 with a substitution or deletion at theamino acid corresponding to position 423 of SEQ ID NO:1, where thesubstituted amino acid is not R, and wherein the polypeptide haschondrogenic activity. In other embodiments, the polypeptide comprisesamino acids 240-454 of SEQ ID NO:1, amino acids 241-455 of SEQ ID NO:1,or amino acids 242-455 of SEQ ID NO:1, each polypeptide with asubstitution or deletion at the amino acid corresponding to position 423of SEQ ID NO:1, where the substituted amino acid is Q or S.

In some embodiments, a protease-resistant ANGPTL3 polypeptide of theinvention comprises an amino acid sequence having at least 95%, or atleast 96%, at least 97%, at least 98%, or at least 99% identity to aminoacids amino acids 242-455 or 242-460 of SEQ ID NO:1; 241-455 or 241-460of SEQ ID NO:1; amino acids 233-455 or 233-460 of SEQ ID NO:1; aminoacids 228-455 or 228-460 of SEQ ID NO:1, amino acids 226-455- or 226-260of SEQ ID NO:1, or amino acids 225-455- or 225-260 of SEQ ID NO:1 inwhich an amino acid is substituted for an R or K, or an R or K isdeleted. In some embodiments, the substitution or deletion is atposition 423. In some embodiments, a substitution is a polar amino acid,e.g., H, N, Q, S, T, A, or Y. In some embodiments, a substitution is H,N, Q, S, T, or Y. In some embodiments, the substitution is S or Q. Insome embodiments, the substitution is a Q. In certain embodiments thereis a deleted residue at position 423 relative to SEQ ID NO:1.

In some embodiments, a protease-resistant ANGPTL3 polypeptide of theinvention is 250 or 240 or fewer amino acids in length and comprises theamino acid sequence of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ IDNO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69, and SEQ ID NO:70. In some embodiments, a protease-resistantANGPTL3 polypeptide of the invention is 230 or 225 or fewer amino acidsin length and comprises the amino acid sequence of SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:68, SEQ IDNO:69, or SEQ ID NO:70.

In some embodiments the protease resistant ANGPTL3 proteins of theinvention comprise an amino acid sequence having at least 95% identity,or at least 96%, 97%, 98%, or 99% identity, to the C-terminal canine,bovine, or equine ANGPTL3 protein sequence. In some embodiments, theprotease-resistant ANGPTL3 proteins of the invention comprise at least afragment (e.g., at least 100, 150, 200, 215 contiguous amino acids) of anative canine (SEQ ID NO:4), equine (SEQ ID NO:5), or bovine (SEQ IDNO:6) ANGPTL3 protein sequence, or a substantially identical sequence tothe native canine, bovine, or equine ANGPTL3 protein sequence whereinthe polypeptide comprises an amino acid that is a polar amino acid otherthan K or R at position 423 or the polypeptide comprises a deletion atposition 423, as determined with reference to SEQ ID NO:1, and thepolypeptide has chondrogenic activity. In some embodiments, an isolatedpolypeptide comprises an amino acid sequence having at least 95%identity, or at least 96%, 97%, 98%, or 99% identity, to SEQ ID NO:42 orSEQ ID NO:43, wherein the polypeptide comprises an amino acid that is apolar amino acid other than K or R at position 423 or the polypeptidecomprises a deletion at position 423, as determined with reference toSEQ ID NO:1, and the polypeptide has chondrogenic activity. In someembodiments, a polypeptide has at least 95% identity, or at least or atleast 96%, 97%, 98%, or 99% identity, to SEQ ID NO:42, or SEQ ID NO:43wherein the polypeptide comprises an amino acid that is a polar aminoacid other than K or R at position 423 or the polypeptide comprises adeletion at position 423, as determined with reference to SEQ ID NO:1,and the polypeptide has chondrogenic activity. In certain embodiments apolypeptide comprises SEQ ID NO:42, or SEQ ID NO:43. In a furtherembodiment, a polypeptide is SEQ ID NO:42, or SEQ ID NO:43.

In some embodiments, a protease-resistant ANGPTL3 of the inventioncomprises an amino acid sequence that has at least 95%, or at least 96%,97%, 98%, or at least 99% identity to amino acids 232-454 of SEQ IDNO:4, amino acids 240-454 of SEQ ID NO:4, amino acids 227-454 of SEQ IDNO:4, or amino acids 224-454 of SEQ ID NO:4 in which an amino acid issubstituted for an R or K or there is a deletion of an R or K. In someembodiments, the substitution or deletion is at position 422 of SEQ IDNO:4, which corresponds to position 423 of SEQ ID NO:1. In someembodiments, a substitution is a polar amino acid, e.g., H, N, Q, S, T,A, or Y. In some embodiments, a substitution is H, N, Q, S, T, or Y. Insome embodiments, the substitution is S or Q. In some embodiments, thesubstitution is a Q. In some embodiments an amino acid deletion is atposition 422 of SEQ ID NO:4.

In some embodiments, a protease-resistant ANGPTL3 of the inventioncomprises an amino acid sequence that has at least 95%, or at least 96%,97%, 98%, or at least 99% identity to amino acids 233-455 of SEQ IDNO:5, amino acids 241-455 of SEQ ID NO:5, amino acids 228-455 of SEQ IDNO:5, or amino acids 225-455 of SEQ ID NO:5 in which an amino acid issubstituted for an R or K or there is a deletion of an R or K. In someembodiments, the substitution or deletion is at position 423 of SEQ IDNO:5, which corresponds to position 423 of SEQ ID NO:1. In someembodiments, a substitution is a polar amino acid, e.g., H, N, Q, S, T,A, or Y. In some embodiments, a substitution is H, N, Q, S, T, or Y. Insome embodiments, the substitution is S or Q. In some embodiments, thesubstitution is a Q. In some embodiments an amino acid deletion is atposition 423 of SEQ ID NO:5.

In some embodiments, a protease-resistant ANGPTL3 of the inventioncomprises an amino acid sequence that has at least 95%, or at least 96%,97%, 98%, or at least 99% identity to amino acids 233-455 of SEQ IDNO:6, amino acids 241-455 of SEQ ID NO:6, amino acids 228-455 of SEQ IDNO:6, or amino acids 225-455 of SEQ ID NO:6 in which an amino acid issubstituted for an R or K or there is a deletion of an R or K. In someembodiments, the substitution or deletion is at position 422 of SEQ IDNO:6, which corresponds to position 423 of SEQ ID NO:1. In someembodiments, a substitution is a polar amino acid, e.g., H, N, Q, S, T,A, or Y. In some embodiments, a substitution is H, N, Q, S, T, or Y. Insome embodiments, the substitution is S or Q. In some embodiments, thesubstitution is a Q. In some embodiments an amino acid deletion is atposition 422 of SEQ ID NO:6.

In some embodiments, a protease-resistant ANGPTL3 polypeptide of theinvention comprises contiguous amino acids corresponding to the aminoacid regions: amino acids 240-454 of SEQ ID NO:4; amino acids 232-454 ofSEQ ID NO:4; amino acids 227-454 of SEQ ID NO:4, or amino acids 224-454of SEQ ID NO:4 in which an amino acid is substituted for an R or K orthere is a deletion of an R or K. In some embodiments, the substitutionor deletion is at position 422 of SEQ ID NO:4 (which is position 423 asdetermined with reference to SEQ ID NO:1). In some embodiments, asubstitution is a polar amino acid, e.g., H, N, Q, S, T, A, or Y. Insome embodiments, a substitution is H, N, Q, S, T, or Y. In someembodiments, the substitution is S or Q. In some embodiments, thesubstitution is Q. In some embodiments an amino acid deletion is atposition 422 of SEQ ID NO:4.

In some embodiments, a protease-resistant ANGPTL3 polypeptide of theinvention comprises contiguous amino acids corresponding to the aminoacid regions: amino acids 241-455 of SEQ ID NO:5; amino acids 233-455 ofSEQ ID NO:5; amino acids 228-455 of SEQ ID NO:5, or amino acids 225-455of SEQ ID NO:5 in which an amino acid is substituted for an R or K orthere is a deletion of an R or K. In some embodiments, the substitutionor deletion is at position 423 (which corresponds to position 423 asdetermined with reference to SEQ ID NO:1). In some embodiments, asubstitution is a polar amino acid, e.g., H, N, Q, S, T, A, or Y. Insome embodiments, a substitution is H, N, Q, S, T, or Y. In someembodiments, the substitution is S or Q. In some embodiments, thesubstitution is Q. In some embodiments an amino acid deletion is atposition 423 of SEQ ID NO:5.

In some embodiments, a protease-resistant ANGPTL3 polypeptide of theinvention comprises contiguous amino acids corresponding to the aminoacid regions: amino acids 241-455 of SEQ ID NO:6; amino acids 233-455 ofSEQ ID NO:6; amino acids 228-455 of SEQ ID NO:6, or amino acids 225-455of SEQ ID NO:6 in which an amino acid is substituted for an R or K orthere is a deletion of an R or K. In some embodiments, the substitutionor deletion is at position 422 of SEQ ID NO:6 (which is position 423 asdetermined with reference to SEQ ID NO:1). In some embodiments, asubstitution is a polar amino acid, e.g., H, N, Q, S, T, A, or Y. Insome embodiments, a substitution is H, N, Q, S, T, or Y. In someembodiments, the substitution is S or Q. In some embodiments, thesubstitution is Q. In some embodiments there is a deletion at position422 of SEQ ID NO:6.

The ANGPTL3 proteins of the invention as described above may includenative ANGPTL3 protein sequences flanking the regions described above.Alternatively, in some embodiments, the ANGPTL3 proteins of theinvention can include non-native ANGPTL3 protein flanking sequences. Forexample, the chondrogenic active portion of an ANGPTL3 protein can befused to one or more fusion partners and/or heterologous amino acids toform a fusion protein. Fusion partner sequences can include, but are notlimited to, amino acid tags, non-L (e.g., D-) amino acids or other aminoacid mimetics to extend in vivo half-life and/or protease resistance,targeting sequences or other sequences.

In some embodiments, a polypeptide of the invention is PEGylated. Insome embodiments, a polypeptide of the invention is fused to aheterologous peptide. In certain embodiments a polypeptide is fused toany one of human serum albumin (HSA), an immunoglobulin heavy chainconstant region (Fc), a polyhistidine, a glutathione S transferase(GST), a thioredoxin, a protein A, a protein G, a maltose bindingprotein (MBP), or a fragment of any of the foregoing heterologouspolypeptide(s). In particular embodiments a heterologous polypeptide isfused at the amino-terminal end of the polypeptide of the invention. Inadditional or alternative embodiments a heterologous polypeptide isfused at the carboxy-terminal end of the polypeptide of the invention.

ANGPTL3 proteins of the invention have chondrogenic activity and areprotease-resistant. As defined herein, chondrogenesis or chondrogenicactivity refers to the development of chondrocytes from MSCs. Indicatorsof chondrogenic activity include, but are not limited to, cartilagematrix production. Cartilage matrix production may be measured byvarious markers, for example, such as Sox9, type II collagen, orglycosaminoglycan (GAG) production. In some embodiments, GAG productionis measured as a marker for cartilage matrix production. In someembodiments, a 3-fold increase in GAG production with cartilage specificprotein expression indicates positive cartilage matrix production.

A polypeptide may be evaluated for protease resistance using any knownassay that measures cleavage by a serine protease such as trypsin. Insome embodiments, the protease employed to evaluate proteolysissusceptibility is the serine protease trypsin. A polypeptide isconsidered to be protease-resistant if it has reduced sensitivity totrypsin when compared to its wild-type counterpart. An example of anassay is to measure the amount of cleaved product that is generated whena polypeptide is exposed to trypsin over a period of time in comparisonto a corresponding native human peptide. Cleavage can be measured usingany known assay, e.g., SDS PAGE or LCMS. An illustrative assay isprovided in the Examples section.

In an illustrative assay, limited proteolysis by trypsinolysis isperformed by incubating 10 ng of the protein to be evaluated withtrypsin at mass ratio of 8000:1 (Protein:Trypsin) for 1 hr at roomtemperature. The trypsinolysis reaction can then be quenched by additionof acetic acid to bring the reaction to pH 3.0. The quenched samples arethen separated analyzed by SDS-PAGE, e.g., on a 4-12% Tris-Bis gel toidentify proteins which are resistant to cleavage from those that arecleaved by the appearance of a fragment that is generated by trypsincleavage. The cleavage product is absent or reduced in theprotease-resistant polypeptides in comparison to their wildtypecounterparts.

In some embodiments, the ANGPTL3 polypeptides of the invention willcomprise at least one non-naturally encoded amino acid. In someembodiments, a polypeptide comprises 1, 2, 3, 4, or more unnatural aminoacids. Methods of making and introducing a non-naturally-occurring aminoacid into a protein are known. See, e.g., U.S. Pat. Nos. 7,083,970; and7,524,647. The general principles for the production of orthogonaltranslation systems that are suitable for making proteins that compriseone or more desired unnatural amino acid are known in the art, as arethe general methods for producing orthogonal translation systems. Forexample, see International Publication Numbers WO 2002/086075, entitled“METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONALtRNA-AMINOACYL-tRNA SYNTHETASE PAIRS;” WO 2002/085923, entitled “IN VIVOINCORPORATION OF UNNATURAL AMINO ACIDS;” WO 2004/094593, entitled“EXPANDING THE EUKARYOTIC GENETIC CODE;” WO 2005/019415, filed Jul. 7,2004; WO 2005/007870, filed Jul. 7, 2004; WO 2005/007624, filed Jul. 7,2004; WO 2006/110182, filed Oct. 27, 2005, entitled “ORTHOGONALTRANSLATION COMPONENTS FOR THE VIVO INCORPORATION OF UNNATURAL AMINOACIDS” and WO 2007/103490, filed Mar. 7, 2007, entitled “SYSTEMS FOR THEEXPRESSION OF ORTHOGONAL TRANSLATION COMPONENTS IN EUBACTERIAL HOSTCELLS.” For discussion of orthogonal translation systems thatincorporate unnatural amino acids, and methods for their production anduse, see also, Wang and Schultz, (2005) “Expanding the Genetic Code.”Angewandte Chemie Int Ed 44: 34-66; Xie and Schultz, (2005) “AnExpanding Genetic Code.” Methods 36: 227-238; Xie and Schultz, (2005)“Adding Amino Acids to the Genetic Repertoire.” Curr Opinion in ChemicalBiology 9: 548-554; and Wang, et al., (2006) “Expanding the GeneticCode.” Annu Rev Biophys Biomol Struct 35: 225-249; Deiters, et al,(2005) “In vivo incorporation of an alkyne into proteins in Escherichiacoli.” Bioorganic & Medicinal Chemistry Letters 15:1521-1524; Chin, etal., (2002) “Addition of p-Azido-L-phenylalanine to the Genetic Code ofEscherichia coli.” J Am Chem Soc 124: 9026-9027; and InternationalPublication No. WO2006/034332, filed on Sep. 20, 2005. Additionaldetails are found in U.S. Pat. Nos. 7,045,337; 7,083,970; 7,238,510;7,129,333; 7,262,040; 7,183,082; 7,199,222; and 7,217,809.

A “non-naturally encoded amino acid” refers to an amino acid that is notone of the common amino acids or pyrolysine, pyrroline-carboxy-lysine,or selenocysteine. Other terms that may be used synonymously with theterm “non-naturally encoded amino acid” are “non-natural amino acid,”“unnatural amino acid,” “non-naturally-occurring amino acid,” andvariously hyphenated and non-hyphenated versions thereof. The term“non-naturally encoded amino acid” also includes, but is not limited to,amino acids that occur by modification (e.g. post-translationalmodifications) of a naturally encoded amino acid (including but notlimited to, the 20 common amino acids or pyrrolysine,pyrroline-carboxy-lysine, and selenocysteine) but are not themselvesnaturally incorporated into a growing polypeptide chain by thetranslation complex. Examples of such non-naturally-occurring aminoacids include, but are not limited to, N-acetylglucosaminyl-L-serine,N-acetylglucosaminyl-L-threonine, and 0-phosphotyrosine.

A non-naturally encoded amino acid is typically any structure having anysubstituent side chain other than one used in the twenty natural aminoacids. Because the non-naturally encoded amino acids of the inventiontypically differ from the natural amino acids only in the structure ofthe side chain, the non-naturally encoded amino acids form amide bondswith other amino acids, including but not limited to, natural ornon-naturally encoded, in the same manner in which they are formed innaturally occurring polypeptides. However, the non-naturally encodedamino acids have side chain groups that distinguish them from thenatural amino acids. For example, R optionally comprises an alkyl-,aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-,hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate,boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine,aldehyde, ester, thioacid, hydroxylamine, amino group, or the like orany combination thereof. Other non-naturally occurring amino acids ofinterest that may be suitable for use in the present invention include,but are not limited to, amino acids comprising a photoactivatablecross-linker, spin-labeled amino acids, fluorescent amino acids, metalbinding amino acids, metal-containing amino acids, radioactive aminoacids, amino acids with novel functional groups, amino acids thatcovalently or noncovalently interact with other molecules, photocagedand/or photoisomerizable amino acids, amino acids comprising biotin or abiotin analogue, glycosylated amino acids such as a sugar substitutedserine, other carbohydrate modified amino acids, keto-containing aminoacids, amino acids comprising polyethylene glycol or polyether, heavyatom substituted amino acids, chemically cleavable and/or photocleavableamino acids, amino acids with an elongated side chains as compared tonatural amino acids, including but not limited to, polyethers or longchain hydrocarbons, including but not limited to, greater than about 5or greater than about 10 carbons, carbon-linked sugar-containing aminoacids, redox-active amino acids, amino thioacid containing amino acids,and amino acids comprising one or more toxic moiety.

Exemplary non-naturally encoded amino acids that may be suitable for usein the present invention and that are useful for reactions with watersoluble polymers include, but are not limited to, those with carbonyl,aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactivegroups. In some embodiments, non-naturally encoded amino acids comprisea saccharide moiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occurring N- or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occurring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

Another type of modification that can optionally be introduced into theANGPTL3 proteins of the invention (e.g. within the polypeptide chain orat either the N- or C-terminal), e.g., to extend in vivo half-life, isPEGylation or incorporation of long-chain polyethylene glycol polymers(PEG). Introduction of PEG or long chain polymers of PEG increases theeffective molecular weight of the present polypeptides, for example, toprevent rapid filtration into the urine. In some embodiments, a Lysineresidue in the ANGPTL3 sequence is conjugated to PEG directly or througha linker. Such linker can be, for example, a Glu residue or an acylresidue containing a thiol functional group for linkage to theappropriately modified PEG chain. An alternative method for introducinga PEG chain is to first introduce a Cys residue at the C-terminus or atsolvent exposed residues such as replacements for Arg or Lys residues.This Cys residue is then site-specifically attached to a PEG chaincontaining, for example, a maleimide function. Methods for incorporatingPEG or long chain polymers of PEG are well known in the art (described,for example, in Veronese, F. M., et al., Drug Disc. Today 10: 1451-8(2005); Greenwald, R. B., et al., Adv. Drug Deliv. Rev. 55: 217-50(2003); Roberts, M. J., et al., Adv. Drug Deliv. Rev., 54: 459-76(2002)), the contents of which is incorporated herein by reference.Other methods of polymer conjugations known in the art can also be usedin the present invention. In some embodiments,poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) is introduced as apolymer conjugate with the ANGPTL3 proteins of the invention (see, e.g.,WO2008/098930; Lewis, et al., Bioconjug Chem., 19: 2144-55 (2008)). Insome embodiments, a phosphorylcholine-containing polymer conjugate withthe ANGPTL3 proteins can be used in the present invention. A person ofskill would readily recognize that other biocompatible polymerconjugates can be utilized.

A more recently reported alternative approach for incorporating PEG orPEG polymers through incorporation of non-natural amino acids (asdescribed above) can be performed with the present polypeptides. Thisapproach utilizes an evolved tRNA/tRNA synthetase pair and is coded inthe expression plasmid by the amber suppressor codon (Deiters, A, et al.(2004). Bio-org. Med. Chem. Lett. 14, 5743-5). For example,p-azidophenylalanine can be incorporated into the present polypeptidesand then reacted with a PEG polymer having an acetylene moiety in thepresence of a reducing agent and copper ions to facilitate an organicreaction known as “Huisgen [3+2]cycloaddition.”

In certain embodiments, the present invention also contemplates specificmutations of the ANGPTL3 proteins so as to alter the glycosylation ofthe polypeptide. Such mutations may be selected so as to introduce oreliminate one or more glycosylation sites, including but not limited to,O-linked or N-linked glycosylation sites. In certain embodiments, theANGPTL3 proteins of the present invention have glycosylation sites andpatterns unaltered relative to the naturally-occurring ANGPTL3 proteins.In certain embodiments, a variant of ANGPTL3 proteins includes aglycosylation variant wherein the number and/or type of glycosylationsites have been altered relative to the naturally-occurring ANGPTL3proteins. In certain embodiments, a variant of a polypeptide comprises agreater or a lesser number of N-linked glycosylation sites relative to anative polypeptide. An N-linked glycosylation site is characterized bythe sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residuedesignated as X may be any amino acid residue except proline. Thesubstitution of amino acid residues to create this sequence provides apotential new site for the addition of an N-linked carbohydrate chain.Alternatively, substitutions which eliminate this sequence will removean existing N-linked carbohydrate chain. In certain embodiments, arearrangement of N-linked carbohydrate chains is provided, wherein oneor more N-linked glycosylation sites (typically those that are naturallyoccurring) are eliminated and one or more new N-linked sites arecreated.

Exemplary ANGPTL3 proteins variants include cysteine variants whereinone or more cysteine residues are deleted from or substituted foranother amino acid (e.g., serine) relative to the amino acid sequence ofthe naturally-occurring ANGPTL3 proteins. In certain embodiments,cysteine variants may be useful when ANGPTL3 proteins must be refoldedinto a biologically active conformation such as after the isolation ofinsoluble inclusion bodies. In certain embodiments, cysteine variantshave fewer cysteine residues than the native polypeptide. In certainembodiments, cysteine variants have an even number of cysteine residuesto minimize interactions resulting from unpaired cysteines.

In some embodiments, functional variants or modified forms of theANGPTL3 proteins include fusion proteins of an ANGPTL3 protein of theinvention and one or more fusion domains. Well known examples of fusiondomains include, but are not limited to, polyhistidine, Glu-Glu,glutathione S transferase (GST), thioredoxin, protein A, protein G, animmunoglobulin heavy chain constant region (Fc), maltose binding protein(MBP), and/or human serum albumin (HSA). A fusion domain or a fragmentthereof may be selected so as to confer a desired property. For example,some fusion domains are particularly useful for isolation of the fusionproteins by affinity chromatography. For the purpose of affinitypurification, relevant matrices for affinity chromatography, such asglutathione-, amylase-, and nickel- or cobalt-conjugated resins areused. Many of such matrices are available in “kit” form, such as thePharmacia GST purification system and the QLAexpress™ system (Qiagen)useful with (HIS₆) fusion partners. As another example, a fusion domainmay be selected so as to facilitate detection of the ANGPTL3 proteins.Examples of such detection domains include the various fluorescentproteins (e.g., GFP) as well as “epitope tags,” which are usually shortpeptide sequences for which a specific antibody is available. Well knownepitope tags for which specific monoclonal antibodies are readilyavailable include FLAG, influenza virus haemagglutinin (HA), and c-myctags. In some cases, the fusion domains have a protease cleavage site,such as for Factor Xa or Thrombin, which allows the relevant protease topartially digest the fusion proteins and thereby liberate therecombinant proteins therefrom. The liberated proteins can then beisolated from the fusion domain by subsequent chromatographicseparation. In certain embodiments, an ANGPTL3 protein is fused with adomain that stabilizes the ANGPTL3 protein in vivo (a “stabilizer”domain). By “stabilizing” is meant anything that increases serum halflife, regardless of whether this is because of decreased destruction,decreased clearance by the kidney, or other pharmacokinetic effect.Fusions with the Fc portion of an immunoglobulin are known to conferdesirable pharmacokinetic properties on a wide range of proteins.Likewise, fusions to human serum albumin can confer desirableproperties. Other types of fusion domains that may be selected includemultimerizing (e.g., dimerizing, tetramerizing) domains and functionaldomains (that confer an additional biological function, as desired).Fusions may be constructed such that the heterologous peptide is fusedat the amino terminus of a polypeptide of the invention and/or at thecarboxy terminus of a polypeptide of the invention.

Nucleic Acids Encoding Angiopoietin-Like 3 Protease-ResistantPolypeptides

The invention also provides nucleic acids encoding protease resistantpolypeptides of the invention and expression vectors and host cells forexpression of a protease resistant polypeptide. In other aspects, theinvention provides a polynucleotide encoding a polypeptide of theinvention and expression vectors and host cells comprising such apolynucleotide. In some embodiments, the polynucleotide is optimized forexpression in the host cells. In some embodiments, the inventionprovides a method of ameliorating or preventing arthritis or jointinjury in a human patient, the method comprising: administering to ajoint of the patient an expression vector encoding a polypeptide of theinvention whereupon expression of the polypeptide ameliorates orprevents arthritis or joint injury in the patient. In some embodiments,the patient has arthritis or joint injury. In some embodiments, theindividual does not have, but is at risk for, arthritis or joint injury.In some embodiments, the arthritis is osteoarthritis, trauma arthritis,or autoimmune arthritis.

Expressing polypeptides of the invention employs routine techniques inthe field of recombinant genetics. Basic texts disclosing the generalmethods of use in this invention include Sambrook and Russell eds.(2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the seriesAusubel et al. eds. (2007 with updated through 2010) Current Protocolsin Molecular Biology, among others known in the art.

Expression can employ any appropriate host cells known in the art, forexample, mammalian host cells, bacterial host cells, yeast host cells,insect host cells, etc. Both prokaryotic and eukaryotic expressionsystems are widely available. In some embodiments, the expression systemis a mammalian cell expression, such as a CHO cell expression system. Insome embodiments, a nucleic acid may be codon-optimized to facilitateexpression in a desired host cell.

Nonviral vectors and systems include plasmids and episomal vectors,typically comprising an expression cassette for expressing a protein orRNA, and human artificial chromosomes (see, e.g., Harrington et al., NatGenet 15:345, 1997). For example, nonviral vectors useful for expressionof the polypeptides of the invention in mammalian (e.g., human) cellsinclude pThioHis A, B & C, pcDNA3. I/His, pEBVHis A, B & C (Invitrogen,San Diego, Calif.), MPSV vectors, and numerous other vectors known inthe art for expressing other proteins. Useful viral vectors include, butare not limited to, vectors based on adenoviruses, adenoassociatedviruses, herpes viruses, vectors based on SV40, papilloma virus, HBPEpstein Barr virus, fowpox vectors, vaccinia virus vectors and SemlikiForest virus (SFV).

The choice of expression vector depends on the intended host cells inwhich the vector is to be expressed. Typically, the expression vectorscontain a promoter and other regulatory sequences (e.g., enhancers) thatare operably linked to the polynucleotides encoding a polypeptide of theinvention. In some embodiments, an inducible promoter is employed toprevent expression of inserted sequences except under inducingconditions. Inducible promoters include, e.g., arabinose, lacZ, ametallothionein promoter, a glucocorticoid promoters or a heat shockpromoter. In addition, other regulatory elements may also beincorporated to improve expression of a nucleic acid encoding apolypeptide of the invention, e.g., enhancers, ribosomal binding site,transcription termination sequences, and the like.

In some embodiments, a nucleic acid encoding an polypeptide of theinvention may also include a sequence encoding a secretion signalsequence so that the polypeptide is secreted from the host cell. Such asequence can be provided by the vector, or as part of the ANGPTL3nucleic acid that is present in the vector.

Methods for introducing expression vectors containing the polynucleotidesequences of interest vary depending on the type of cellular host. Forexample, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for other cellular hosts (see generallySambrook et al., supra). Other methods include, e.g., electroporation,calcium phosphate treatment, liposome-mediated transformation, injectionand microinjection, ballistic methods, virosomes, immunoliposomes,polycation: nucleic acid conjugates, naked DNA, artificial virions,fusion to the herpes virus structural protein VP22, agent-enhanceduptake of DNA, and ex vivo transduction. For long-term, high-yieldproduction of recombinant proteins, stable expression will often bedesired. For example, cell lines which stably express polypeptides ofthe invention can be prepared using expression vectors of the inventionwhich contain viral origins of replication or endogenous expressionelements and a selectable marker gene.

In some embodiments, nucleic acids encoding protease resistant ANGPTL3polypeptides of the invention can be delivered to a patient fortreatment of a joint-related injury or disease. Delivery of such nucleicacids can be achieved using any means known in the art, but is typicallyperformed using direct injection into the affected joint. In someembodiments, a DNA is delivered as naked DNA using direct injection intothe joint. In some embodiments, a viral vector is employed, including,but not limited to, an adenovirus or adenovirus-associated vector, aherpes virus vector, fowlpox virus, or a vaccinia virus vector.

Methods of Therapeutic Use of Polypeptides, and Indications

Provided methods of the invention include a method of treating a subjectcomprising administering to the subject a therapeutically effectiveamount of a polypeptide of the invention, wherein the subject has or isat risk of joint damage or arthritis. The invention also provides amethod of ameliorating or preventing arthritis or joint injury in ahuman patient, the method comprising: administering to a joint of thepatient a composition comprising an effective amount of a polypeptide ofthe invention, thereby ameliorating or preventing arthritis or jointinjury in the patient. In some embodiments, the patient has arthritis orjoint injury. In some embodiments, the individual does not have, but isat risk for, arthritis or joint injury. In some embodiments, thearthritis is osteoarthritis, trauma arthritis, or autoimmune arthritis.In some embodiments, the composition administered to the furthercomprises hyaluronic acid.

In another aspect, the invention provides a method of inducingdifferentiation of mesenchymal stem cells into chondrocytes, the methodcomprising, contacting mesenchymal stem cells with a sufficient amountof a polypeptide of the invention to induce differentiation of the stemcells into chondrocytes. In some embodiments, the method is performed invivo and the stem cells are present in a human patient.

It is contemplated that polypeptides, compositions, and methods of thepresent invention may be used to treat, ameliorate or prevent any typeof articular cartilage damage (e.g., joint damage or injury) including,for example, damage arising from a traumatic event or tendon or ligamenttear. In some embodiments, proteins of the invention are administered toprevent or ameliorate arthritis or joint damage, for example where thereis a genetic or family history of arthritis or joint damage or jointinjury or prior or during joint surgery. In some embodimentspolypeptides, compositions and methods are used to treat joint damage.In particular embodiments joint damage is traumatic joint injury. Inother embodiments joint damage is damage arising from age or inactivity.In yet other embodiments joint damage is damage arising from anautoimmune disorder. In some embodiments of the invention, polypeptides,compositions, and methods of the present invention may be used to treat,ameliorate or prevent osteoarthritis. In some embodiments, thepolypeptides, compositions and methods are used to ameliorate or preventarthritis in a subject at risk of having or acquiring arthritis. In someembodiments, the polypeptides, compositions and methods are used toameliorate or prevent joint damage in a subject at risk of having oracquiring joint damage.

In some embodiments, polypeptides, compositions, and methods of thepresent invention provide a method for stimulating chondrocyteproliferation and cartilage production in cartilagenous tissues thathave been damaged, e.g., due to traumatic injury or chondropathy. Inparticular embodiments polypeptides, compositions, and methods of thepresent invention are useful for treatment of cartilage damage injoints, e.g., at articulated surfaces, e.g., spine, shoulder, elbow,wrist, joints of the fingers, hip, knee, ankle, and joints of the feet.Examples of diseases or disorders that may benefit from treatmentinclude osteoarthritis, rheumatoid arthritis, other autoimmune diseases,or osteochondritis dessicans. In addition, cartilage damage ordisruption occurs as a result of certain genetic or metabolic disorders,cartilage malformation is often seen in forms of dwarfism in humans,and/or cartilage damage or disruption is often a result ofreconstructive surgery; thus polypeptides, compositions, and methodswould be useful therapy in these patients, whether alone or inconnection with other approaches.

It is further contemplated that polypeptides, compositions, and methodsof the present invention may be used to treat, ameliorate or preventvarious cartilagenous disorders and/or associated symptoms or effects ofsuch conditions. Exemplary conditions or disorders for treatment,amelioration and/or prevention with polypeptides, compositions, andmethods of the invention, include, but are not limited to systemic lupuserythematosis, rheumatoid arthritis, juvenile chronic arthritis,osteoarthritis, degenerative disc disease, spondyloarthropathies, EhlersDanlos syndrome, systemic sclerosis (scleroderma) or tendon disease.Other conditions or disorders that may benefit from treatment withpolypeptides for amelioration of associated effects include idiopathicinflammatory myopathies (dermatomyositis, polymyositis), Sjögren'ssyndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia(immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barr syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease (ulcerative colitis: Crohn's disease),gluten-sensitive enteropathy, and Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft-versus-host-disease.

A “patient” as used herein refers to any subject that is administered atherapeutic polypeptide of the invention. It is contemplated that thepolypeptides, compositions, and methods of the present invention may beused to treat a mammal. As used herein a “subject” refers to any mammal,including humans, domestic and farm animals, and zoo, sports or petanimals, such as cattle (e.g. cows), horses, dogs, sheep, pigs, rabbits,goats, cats, etc. In some embodiments of the invention, the subject is ahuman. In certain embodiments, the subject is a horse. In otherembodiments the subject is a dog.

In some embodiments, the polypeptides of the invention can beheterologous to the mammal to be treated. For example, a human ANGPTL3protein or fragments thereof, a protein or peptide derived from a humanANGPTL3 protein (e.g., a modified human ANGPTL3 protein, a conservativevariant of human ANGPTL3 protein, a peptidomimetic derived from a humanANGPTL3 protein) are used in the treatment of an animal such as anequine, bovine or canine. In some embodiments, a heterologous ANGPTL3protein can be used to expand chondrocyte populations in culture fortransplantation. In some embodiments, expanded cultures will then beoptionally admixed with polypeptides and compositions homologous to themammal to be treated, and placed in the joint space or directly into thecartilage defect. Alternatively, polypeptides of the invention arederived from the same species, i.e., a human ANGPTL3 protein orfragments thereof, a protein or peptide derived from a human ANGPTL3protein (e.g., a modified human ANGPTL3 protein, a conservative variantof human ANGPTL3 protein, a peptidomimetic derived from a human ANGPTL3protein) is used in the treatment of a human patient. By using a proteinderived from the same species of mammal as is being treated, inadvertentimmune responses may be avoided.

In some embodiments, polypeptides and compositions of the presentinvention are applied by direct injection into the synovial fluid of ajoint, systemic administration (oral or intravenously) or directly intoa cartilage defect, either alone or complexed with a suitable carrierfor extended release of protein. In some embodiments, polypeptides orcompositions are administered in a biocompatible matrix or scaffold.Polypeptides, compositions, and methods of the present invention canalso be used in conjunction with a surgical procedure at an affectedjoint. Administration of a polypeptide of the invention may occur priorto, during or in conjunction with, and/or after a surgical procedure.For example, polypeptides, compositions and methods of the invention canbe used to expand chondrocyte populations in culture for autologous orallogenic chondrocyte implantation (ACI). Chondrocytes can be optionallyimplanted with concurrent treatment consisting of administration ofpolypeptides and compositions of the present invention. In theseprocedures, for example, chondrocytes can be harvested arthroscopicallyfrom an uninjured minor load-bearing area of a damaged joint, and can becultured in vitro, optionally in the presence of polypeptides andcompositions of the present invention and/or other growth factors toincrease the number of cells prior to transplantation. Expanded culturesare then optionally admixed with polypeptides and compositions of thepresent invention and/or placed in the joint space or directly into thedefect. In certain embodiments, expanded cultures (optionally withpolypeptides of the present invention) are placed in the joint spacesuspended in a matrix or membrane. In other embodiments, polypeptidesand compositions of the present invention can be used in combinationwith one or more periosteal or perichondrial grafts that containcartilage forming cells and/or help to hold the transplantedchondrocytes or chondrocyte precursor cells in place. In someembodiments, polypeptides and compositions of the present invention areused to repair cartilage damage in conjunction with other procedures,including but not limited to lavage of a joint, stimulation of bonemarrow, abrasion arthroplasty, subchondral drilling, or microfracture ofproximal subchondral bone. Optionally, following administration ofpolypeptides and compositions of the present invention and growth ofcartilage, additional surgical treatment may be beneficial to suitablycontour newly formed cartilage surface(s).

Pharmaceutical Compositions

Therapeutic compositions comprising provided polypeptides are within thescope of the present invention, and are specifically contemplated inlight of the identification of several polypeptide sequences exhibitingenhanced stability and protease resistance. Thus, in a further aspect,the invention provides a pharmaceutical composition comprising atherapeutically effective amount of a polypeptide of the invention. Incertain embodiments, pharmaceutical compositions further comprise apharmaceutically or physiologically acceptable carrier. In someembodiments, a pharmaceutical composition further comprises a hyaluronicacid or a derivative thereof.

In addition, the invention provides a method of ameliorating orpreventing arthritis or joint injury in a human patient, the methodcomprising: administering to a joint of the patient a compositioncomprising an effective amount of a polypeptide of the invention,thereby ameliorating or preventing arthritis or joint injury in thepatient. In some embodiments, the patient has arthritis or joint injury.In some embodiments, the individual does not have, but is at risk for,arthritis or joint injury. In some embodiments, the arthritis isosteoarthritis, trauma arthritis, or autoimmune arthritis. In someembodiments, the composition administered to the further compriseshyaluronic acid.

In another aspect, the invention provides a method of inducingdifferentiation of mesenchymal stem cells into chondrocytes, the methodcomprising, contacting mesenchymal stem cells with a sufficient amountof a polypeptide of the invention to induce differentiation of the stemcells into chondrocytes. In some embodiments, the method is performed invivo, the stem cells are present in a human patient, and the contactingcomprises administering to a joint of the patient a compositioncomprising an effective amount of a polypeptide of the invention,thereby inducing differentiation of stem cells into chondrocytes, andgeneration of cartilage.

Therapeutic compositions comprising nucleic acids encoding polypeptidesof the invention can be delivered to a patient for treatment of ajoint-related injury or disease, and are also within the scope of thepresent invention. In some embodiments, pharmaceutical compositionscomprise naked DNA encoding a polypeptide of the invention. In someembodiments, a viral vector is employed to effect delivery and apharmaceutical composition comprises a vector encoding a polypeptide ofthe invention, including, but not limited to, an adenovirus oradenovirus-associated vector, a herpes virus vector, fowlpox virus, or avaccinia virus vector. Pharmaceutical compositions comprise atherapeutically effective amount of a nucleic acid encoding apolypeptide of the invention with a pharmaceutically or physiologicallyacceptable carrier.

In another aspect of the present invention, provided polypeptides foruse as a medicament for treatment of joint damage is contemplated. Incertain embodiments polypeptides of the invention for use as amedicament for amelioration of arthritis or joint damage are provided.In some embodiments arthritis is osteoarthritis, trauma arthritis orautoimmune arthritis. In some embodiments joint damage is traumaticjoint injury, autoimmune damage, age related damage, or damage relatedto inactivity. In other embodiments, nucleic acid encoding a polypeptideof the invention for use in a medicament is provided.

Formulations suitable for administration include excipients, includingbut not limited to, aqueous and non-aqueous solutions, isotonic sterilesolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic, and aqueous andnon-aqueous sterile suspensions that can include suspending agents,solubilizers, thickening agents, stabilizers, and preservatives. Incertain embodiments pharmaceutical compositions comprise atherapeutically effective amount of a peptide in admixture with apharmaceutically acceptable formulation agent selected for suitabilitywith the mode of administration, delivery format, and desired dosage.See, e.g., Remington's Pharmaceutical Sciences (18th Ed., A. R. Gennaro,ed., Mack Publishing Company 1990), and subsequent editions of the same.The primary vehicle or carrier in a pharmaceutical composition can beaqueous or non-aqueous in nature. For example, a suitable vehicle orcarrier for injection can be water, physiological saline solution orartificial cerebrospinal fluid, optionally supplemented with othermaterials common in compositions for parenteral administration. Forexample, buffers may be used, e.g., to maintain the composition atphysiological pH or at a slightly lower pH, typically within a range offrom about pH 5 to about pH 8, and may optionally include sorbitol,serum albumin, detergent, or other additional component. In certainembodiments pharmaceutical compositions comprising polypeptides or anucleic acid encoding a polypeptide of the invention can be prepared forstorage in a lyophilized form using appropriate excipients (e.g.,sucrose).

In yet other embodiments formulation with an agent, such as injectablemicroshperes, bio-erodable particles, polymeric compounds, beads, orliposomes or other biocompatible matrix that provides for controlled orsustained release of the polypeptide or a nucleic acid encoding apolypeptide of the invention can then be delivered via a depotinjection. For example, polypeptides or nucleic acid encoding apolypeptide of the invention may be encapsulated in liposomes, orformulated as microparticles or microcapsules or may be incorporatedinto other vehicles, such as biodegradable polymers, hydrogels,cyclodextrins (see for example Gonzalez et al., 1999, BioconjugateChem., 10, 1068-1074; Wang et al., International PCT publication Nos. WO03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCAmicrospheres (see for example U.S. Pat. No. 6,447,796 and US PatentApplication Publication No. US 2002130430), biodegradable nanocapsules,and bioadhesive microspheres, or by proteinaceous vectors (O'Hare andNormand, International PCT Publication No. WO 00/53722) or by the use ofconjugates. Still other suitable delivery mechanisms include implantabledelivery devices.

The dose of a compound of the present invention for treating theabove-mentioned diseases or disorders varies depending upon the mannerof administration, the age and/or the body weight of the subject, andthe condition of the subject to be treated, and ultimately will bedecided by the attending physician or veterinarian. The doseadministered to a subject, in the context of the present inventionshould be sufficient to effect a beneficial response in the subject overtime. Such a dose is a “therapeutically effective amount”. Accordingly,an appropriate dose may be determined by the efficacy of the particularprotein or a nucleic acid encoding a polypeptide of the inventionemployed and the condition of the subject, as well as the body weight orsurface area of the area to be treated. The size of the dose also willbe determined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular proteinor vector in a particular subject. Administration can be accomplishedvia single or divided doses, or as a continuous infusion via animplantation device or catheter. Frequency of dosing will depend uponthe pharmacokinetic parameters of the polypeptide or a nucleic acidencoding a polypeptide of the invention in the formulation used. Aclinician may titer dosage and/or modify administration to achieve thedesired therapeutic effects. A typical dosage ranges from about 0.01μg/kg to about 100 mg/kg, depending on the factors. In certainembodiments, a dosage ranges from about 0.1 μg/kg up to about 10 mg/kg;or about 0.1 μg/kg; about 0.5 μg/kg; about 1 μg/kg; about 2 μg/kg; about.5 μg/kg; about 10 μg/kg; about 15 μg/kg; about 20 μg/kg; about 25μg/kg; about 30 μg/kg; about 35 μg/kg; about 40 μg/kg; about 45 μg/kg;about 50 μg/kg; about 55 μg/kg; about 60 μg/kg; about 65 μg/kg; about 75μg/kg; about 85 μg/kg; about 100 μg/kg. In certain embodiments a dosageis about 50 μg/kg; about 100 μg/kg; about 150 μg/kg; about 200 μg/kg;about 250 μg/kg; about 300 μg/kg; about 350 μg/kg; about 400 μg/kg;about 450 μg/kg; about 500 μg/kg; about 550 μg/kg; about 600 μg/kg;about 650 μg/kg; about 700 μg/kg; about 750 μg/kg; about 800 μg/kg;about 850 μg/kg; about 900 μg/kg; about 950 μg/kg; about 1 mg/kg; about2 mg/kg; about 3 mg/kg; about 4 mg/kg; about 5 mg/kg; about 6 mg/kg;about 7 mg/kg; about 8 mg/kg; about 9 mg/kg; about 10 mg/kg.

Methods of Administration

Any method for delivering the proteins or a nucleic acid encoding apolypeptide of the invention of the invention to an affected joint canbe used. In the practice of this invention, compositions can beparenterally administered, for example injected, e.g., intra-articularly(i.e., into a joint), intravenously, intramuscularly, subcutaneously;infused, or implanted, e.g., in a membrane, matrix, device, etc. Wheninjected, infused or implanted, delivery can be directed into thesuitable tissue or joint, and delivery may be direct bolus delivery orcontinuous delivery. In some embodiments delivery can be in a suitabletissue located in close proximity to an affected joint. In someembodiments delivery may be via diffusion, or via timed release bolus.In some embodiments, a controlled release system (e.g., a pump) can beplaced in proximity of the therapeutic target, e.g., the joint to whichthe polypeptide is administered. In other embodiments, compositions canbe selected for ingestion, e.g., inhalation or oral delivery.

The therapeutic polypeptides or a nucleic acid encoding a polypeptide ofthe invention of the present invention can also be used effectively incombination with one or more additional active agents (e.g., hyaluronicacid or a derivative or salt thereof, growth factor (e.g., FGF18, BMP7),chondrogenic agent (e.g., oral salmon calcitonin, SD-6010 (iNOSinhibitor), vitamin D3 (choliecalciferol), collagen hydrolyzate,rusalatide acetate, avocado soy unsaponifiables (ASU), a compounddescribed in WO2012/129562, kartogenin), a steroid, a non-steroidalanti-inflammatory agent (NSAID), etc.) depending on the desired therapyor effect to improve or enhance the therapeutic effect of either. Thisprocess can involve administering both agents to the patient at the sametime, either as a single composition or pharmacological formulation thatincludes both agents, or by administering two distinct compositions orformulations, wherein one composition includes a polypeptide or apolynucleotide encoding a polypeptide of the invention and the otherincludes the second agent(s). Administration of a therapeuticcomposition comprising a polypeptide or a polynucleotide encoding apolypeptide of the invention can precede or follow administration of thesecond agent by intervals ranging from minutes to weeks.

Formulations of compounds can be stored in sterile vials as a solution,suspension, gel, emulsion, solid, or as a dehydrated or lyophilizedpowder. Formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials. In some embodiments formulationscan be presented in single or multi-chambered pre-filled syringes (e.g.,liquid syringes, lysosyringes). Solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

Also provided are kits comprising the polypeptides or a nucleic acidencoding a polypeptide of the invention of the invention. In oneembodiment provided are kits for producing a single dose administrationunit. The kit comprises a first container comprising a dried polypeptideor a nucleic acid encoding a polypeptide of the invention and a secondcontainer having an aqueous reconstitution formula. In certainembodiments one container comprises a single chamber pre-filled syringe.In other embodiments the containers are encompassed as a multi-chamberedpre-filled syringe

EXEMPLIFICATION

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1: Protease-Resistant Angpt13 Peptide Constructs

Various N-terminal truncation mutants were constructed to removeO-linked glycosylations and facilitate biophysical proteincharacterization. To identify protease-resistant peptides, amino acidsubstitutions were introduced into various positions of human Angpt13peptide fragments corresponding to the C-terminal region of the peptide.FIG. 1 shows positions of mutations in the human Angpt13. Constructswere initially prepared with His tags. The mutant proteins were: 225-460K423Q (225KQ), 225-460 S424T(225ST), 226-460 K423Q (226KQ), 226-460K4235 (226KS), 228-460 K423Q (228KQ), 228-460 S424T (228ST), 233-460K423Q (233KQ), 233-460 K4235 (233KS), 241-460 K423Q (241KQ), 241-460K4235 (241KS), 241-460 Kde1 (241Kde1), 242-460 K423Q (242KQ), 242-460K4235 (242KS) and 242-460 Kde1 (242Kde1).

His-tagged proteins were expressed in HEK Freestyle′ cells and purifiedby Ni-NTA column chromatography. Tag-less C-terminal constructs werealso cloned, purified by previously described method (Gonzalez R et alPNAS 2010). Briefly, target protein with signal sequence (1-16) wascloned in a mammalian expression vector with cytomegalovirus promoter.At 96 h after DNA/PEI transfection in HEK 293 Freestyle (Invitrogen),media containing secreted target protein were harvested and purified byHi-Trap SP column (GE Healthcare). Protein was eluted between 50 mM MES(pH 6.0), 125 mM NaCl to 50 mM MES (pH 6.0), 150 mM NaCl. SDS-PAGEconfirmed that the purified protein was at least 95% pure.

Protease-resistance was assessed as follows. Limited trypsinolysis wasperformed by incubating 10 ng of each prepared protein with trypsin atmass ratio of 8000:1 (Protein:Trypsin) for 1 h r at room temperature.The trypsinolysis reaction was then quenched by addition of acetic acidto bring the reaction to pH 3.0, and quenched samples were analyzed byLC/MS. A 5 min RP HPLC peak corresponding to the mass of the C-terminal43 amino acids (5424-E460) was evident for the respective wild typeprotein constructs. The clip site was at the same site, i.e., betweenK423 and 5424, as observed during full length wild type ANGPLT3 proteinproduction. This peak was absent when the Lys at the clip site wasmutated to Gln. Each of peptide constructs 225KQ, 228KQ, 233KQ, 233KS,241KQ, and 242KQ; and the wildtype 225 peptide were prepared andanalyzed. The peak corresponding to the mass of the C-terminal 43 aminoacids was absent when the Lys at the clip site was mutated to Gln or Serfor each of the constructs, or when the Lys at position 423 was deleted.

Example 2: Integrin Binding Assays

αVβ3 Integrin Prepared peptides 225KQ, 228KQ, 233KQ, 241KQ and 242KQwere tested in vitro for binding to αVβ3 integrin. Briefly, Maxisorpplates were coated with 2 μg/ml Integrin αVβ3, and variousconcentrations of polypeptide construct (indicated) were added. Boundpeptide was detected by the addition of Anti-ANGPTL3 mAb followed byhorseradish peroxidase-conjugated Goat anti-Mouse IgG antibody. Alltested peptides retained or improved integrin binding capacity. EC₅₀ foreach were determined from the binding data, and results are shown inTABLE 2.

TABLE 2 In vitro binding of ANGPTL3 and engineered polypeptideconstructs to Integrins α5β1 integrin EC₅₀ αVβ3 integrin EC₅₀ WT 3.0543.245 242KQ 1.566 3.076 241KQ 2.693 4.032 233KQ 13.83 6.636 228KQ 4.264.051 225KQ 19.89 11.18

α5β1 Integrin

Prepared peptides 225KQ, 228KQ, 233KQ, 241KQ and 242KQ were tested invitro for binding to α5β1 integrin. Plates were coated with 2 μg/ml asdescribed above but with Integrin α5β1, and various concentrations ofpolypeptide construct (indicated) were added, and detection carried outas described above. All tested peptides retained or improved integrinbinding capacity. EC₅₀ for each were determined from the binding data,and results are shown in TABLE 2.

Example 3: Functional Analysis of Constructs

Cell Culture and Differentiation.

Primary human bone marrow derived mesenchymal stem cells (hMSCs) wereFACS sorted and proven to be >98% positive for CD29, CD44, CD166 andCD105 and <0.1% positive for CD45; and cells were used from passages 2-8for experiments. Human cartilage resident MSCs (hCR-MSCs) were derivedfrom human primary articular chondrocytes, which were separated intosingle cells, clonally grown in MSCGM and validated as MSCs throughchondrogenic, osteogenic and adipogenic differentiation. Cells were FACSsorted and proven to be >98% positive for CD166 and CD105. hCR-MSCs werecultured up to 20 passages with no alteration in the cell profile,growth or differentiation rates identified.

Chondrogenesis.

Peptide constructs of the invention were evaluated in physical andfunctional assays to assess chondrogenesis activity.

Engineered constructs provided herein are derived from ANGPTL3 whichbelongs to a family of seven identified ANGPTL proteins that havestructural similarity to the angiopoietins, but lack the ability to bindthe Tie2 receptor and thus have distinct functions. ANGPTL proteinscontain an N-terminal coiled-coil domain (CCD) and a C-terminalfibrinogen-like domain (FLD), and are believed to be tightly regulatedby their microenvironment and interactions with the extracellular matrix(ECM) such as fibronectin and integrins. Conklin et al., Genomics 62(3):477-482 (1999); Goh Y Y, et al., Am J Pathol 177(6): 2791-2803 (2010);Goh Y Y, et al J Biol Chem 285(43): 32999-33009(2010). Sequences forANGPTL family members most closely related to ANGPTL3, ANGPTL1 (fulllength and C-terminal domain) and ANGPTL4 (full length and C-terminaldomain) are provided in Table 3; and FIG. 12 depicts an alignment acrossthe C-terminal domains of these family members. Sequence identitiesacross the extracellular domains and C-terminal domains ANGPTL1,ANGPTL4, as well as other angiopoietin proteins ANGPTL7, ANGPT1 andANGPT2 are provided in Table 5. The C-terminal domain (CT) of ANGPTL3shares 37% sequence identity with CT ANGPTL1 and 40% sequence identitywith CT ANGPTL4.

Cell-based 2D chondrogenesis was induced in vitro and assessed asdescribed previously in Johnson, K., et al., (2012) Science 336, 717.Briefly, primary human bone marrow derived mesenchymal stem cells(hMSCs) were plated in growth media then subsequently changed to achondrogenic stimulation media with and without constructs.

To initially image nodule formation, wells were fixed and stained withRhodamine B where the nodules were easily detected by eye and imagescaptured by light microscopy. To facilitate high throughput imaged-baseddetection and quantification, chondrogenic nodules were stained withNile red which binds non-specifically to collagens. Nile Red stainednodules were quantified on an Acumen eX3 (high content imaging device)by excitation with a 488 laser for rapid detection of the nodules.

TABLE 3 ANGPTL Family Sequences SEQ ID Construct Sequence 71 hANGPTL1MKTFTWTLGVLFFLLVDTGHCRGGQFKIKKINQRRYPRATDGKEEAKKCA 1-491YTFLVPEQRITGPICVNTKGQDASTIKDMITRMDLENLKDVLSRQKREIDVLQLVVDVDGNIVNEVKLLRKESRNMNSRVTQLYMQLLHEIIRKRDNSLELSQLENKILNVTTEMLKMATRYRELEVKYASLTDLVNNQSVMITLLEEQCLRIFSRQDTHVSPPLVQVVPQHIPNSQQYTPGLLGGNEIQRDPGYPRDLMPPPDLATSPTKSPFKIPPVTFINEGPFKDCQQAKEAGHSVSGIYMIKPENSNGPMQLWCENSLDPGGWTVIQKRTDGSVNFFRNWENYKKGFGNIDGEYWLGLENIYMLSNQDNYKLLIELEDWSDKKVYAEYSSFRLEPESEFYRLRLGTYQGNAGDSMMWHNGKQFTTLDRDKDMYAGNCAHFHKGGWWYNACAHSNLNGVWYRGGHYRSKHQDGIFWAEYRGGSYSLRAVQMMIKPID 72 CTFINEGPFKDCQQAKEAGHSVSGIYMIKPENSNGPMQLWCENSLDPGGWTV hANGPTL1IQKRTDGSVNFFRNWENYKKGFGNIDGEYWLGLENIYMLSNQDNYKLLIE 271-491LEDWSDKKVYAEYSSFRLEPESEFYRLRLGTYQGNAGDSMMWHNGKQFTTLDRDKDMYAGNCAHFHKGGWWYNACAHSNLNGVWYRGGHYRSKHQDGIFW AEYRGGSYSLRAVQMMIKPID73 hANGPTL4 MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQ 1-406LGQGLREHAERTRSQLSALERRLSACGSACQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPM AAEAAS 74 CTSRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQ hANGPTL4RRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLR 179-406DWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAAEAAS

TABLE 4 Chondrogenesis of ANGPTL family member proteins Nodule InductionFormation Type II Genbank Protein activity collagen Accession Angptl1Yes Yes NP_004664 Angptl2 No n/a NP_036230 Angptl3 Yes Yes NP_055310Angptl4 Yes No NP_647475 Angptl6 No No NP_114123 Angptl7 No No NP_066969Angpt2 No n/a NP_001138 Angpt1 No n/a NP_004664

Cell-based 2D chondrogenesis was induced in vitro and assessed asdescribed previously in Johnson, K., et al., (2012) Science 336, 717.Briefly, primary human bone marrow derived mesenchymal stem cells(hMSCs) were plated in growth media then subsequently changed to achondrogenic stimulation media with and without constructs, and culturedfor 7 or 14 days. Cells were then fixed with formaldehyde, washed andthen stained using standard immuno-cytochemical techniques to detectprimary cartilage proteins Pro-collagen Type 2A (PIIANP) (FIG. 2A) andType II collagen (FIG. 2B). For detection of type II collagen, cellswere digested with 0.2% Collagenase II (Worthington Biochemical,Lakewood, N.J.) which was added to the permeabilization solution.Immuno-fluorescence for each protein detected was quantified throughhigh content imaging (Image Express Ultra (Molecular Devices, Sunnyvale,Calif.)), using multi-wavelength cell scoring script, and as describedpreviously. See FIG. 2. Aggrecan expression was monitored by preparingcells as follows: briefly, primary hMSCs (5000 cells) were plated in aGriener 384 well plate. After 24 hours the growth media was removed andreplaced with 25 μl of DMEM containing 1% FBS. Protein constructs werethen added to each well at the indicated dose, and cultures were grownat 37° C. for 3 days. The cells were fixed with 10% formalin andsubjected to immunocytochemical methods to detect Aggrecan proteinexpression. Wells were imaged on the ImageXpress Ultra and quantifiedwith the multi-wavelength cell scoring script, n=6/proteinconcentration. Results are exemplified in FIG. 3B relative to control(cells stimulated without construct, diluent alone) for WT wild typeC-terminal (225-460) ANGPTL3, engineered construct 242KQ or 242Kde1 orfull length ANGPTL1, a related family member of the ANGPTL proteins.Similar results were seen with experiments using each of 225WT, 225KQ,226KQ, 228KQ, 233KQ, 241KQ and 242KQ constructs.

Chondrogenesis assays were carried out using assays and methodsdescribed previously and herein for additional ANGPTL related familymembers. Experiments were carried out to examine whether closely relatedproteins confer chondrogenic activity, and if the activity was retainedin the C-terminal end of the protein. ANGPTL1 and ANGPTL4 demonstratedactivity in nodule formation assays; however, only ANGPTL1 showed aninduction of type II collagen in chondrogenesis assays. See Table 4.Results of nodule formation activity and induction of Type II collagenassays are summarized in Table 4. Additional characterization of ANGPTL1is described herein. See other portions of this Example and FIGS. 3-5.

TABLE 5 Sequence homology among human angiopoeitin like family membersSequence identity among human angiopoeitin like family members (ECD orCTD) Family member Family member % Sequence Identity hANGPTL3_17-460hANGPTL4_26-406 32.6 hANGPTL3_17-460 hANGPTL1_24-491 25.7hANGPTL3_17-460 hANGPTL7_27-346 28.1 hANGPTL3_17-460 hANGPT1_23-498 24.1hANGPTL3_17-460 hANGPT2_19-496 23.4 hANGPTL3_241-460 hANGPTL4_179-40640.0 hANGPTL3_241-460 hANGPTL1_271-491 36.8 hANGPTL3_241-460hANGPTL7_122-343 36.4 hANGPTL3_241-460 hANGPT1_277-497 37.3hANGPTL3_241-460 hANGPT2_275-495 36.4

RNA expression analysis was also used to evaluate expression ofcartilage specific proteins. Briefly, qRT-PCR hMSCs were grown in pelletculture (1×10⁶ cells/pellet) for 3, 7, 10, 21 days in serum free DMEM,1×ITS plus constructs (as indicated). Media was replaced every 3 days.Lubricin, Aggrecan, Sox9, IGF1, IFITM1, Osteocalcin and type X collagenmRNA expression were quantified using Roche LightCycler (data pooledfrom 3 experiments performed in duplicate (n=6)). FIG. 3A representsexpression data at Day 10 for 242KQ and 225WT. Gene expression data wassimilar for all genes at days 3, 7 and 21.

Full length ANGPTL3 had been previously shown to have chondrogenesisactivity in both human and mouse mesenchymal stem cells. Constructs weretested for activity in human, mouse, rat and canine mesenchymal stemcells to demonstrate the ability of additional species cross reactivity.CR-MSCs from mouse, rat, human and dog were cultured with constructs asdescribed above for 18 days. Cultures were fixed and stained usingstandard imunnocytochemical techniques to detect the chondrocytespecific protein type II collagen, and type II collagen positive cellswere quantified using high content imaging. Similar fold increase in theamount of type II collagen quantified was confirmed for each species ofcells evaluated.

Chondroprotection.

Peptide constructs were evaluated in functional assays to assesschondroprotective activity.

An ex vivo glycosaminoglycan (GAG) release inhibition assay (anindicator of matrix damage) was performed as described in Johnson, K.,et al., (2012) Science 336, 717-721. Briefly, bovine cartilage wasisolated, punched into symmetric circles and put into organ culture.Slices were treated for 48 hours with 20 ng/ml TNFα and 10 ng/mloncostatin M (OSM) (inflammatory mediators) to induce degradation of thecartilage matrix in the presence or absence of protein constructs toidentify percent inhibition of glycosaminoglycan (GAG) release. Resultsshown in FIG. 4A depict data pooled from 4 donors, n=12 with theengineered constructs as indicated and WT 225-460.

An in vitro nitric oxide (NO) inhibition assay (an indicator ofchondroprotection) was performed as described in Johnson, K., et al.,(2012) Science 336, 717-721. Briefly, primary chondrocytes were treatedfor 48 hrs with protein constructs as indicated. Greiss reaction wasperformed, to determine the effect of constructs on inhibition of NOrelease as Results shown in FIG. 4B depict results with the engineeredconstructs as indicated and WT Cterminal fragment 225-460. Results shownin FIG. 4C depict results with wild type Cterminal ANGPTL1, engineeredANGPTL3 242KQ or control.

Inhibition of Fibrotic Cartilage Formation.

Primary human articular chondrocytes were cultured as described abovewith the addition of ascorbic acid and the presence or absence ofconstructs (indicated) for 14 days to induce hypertrophy and type Xcollagen expression was assessed by immunoflurescence. Results shown inFIG. 5A depict data with constructs 225WT or 242KQ as indicated. Resultsshown in FIG. 5B depict data with wild type C-terminal ANGPTL1,engineered ANGPTL3 242KQ or 242Kde1 or wild type C-terminal ANGPTL3fragment 225-460 as indicated. The presence of wild type or activeconstructs confer an inhibitory effect on formation of fibroticcartilage under hypertrophic conditions, as detected by expression oftype X collagen.

Angiogenesis.

The WT C-terminal domain of the ANGPTL3 protein has been reported tohave angiogenic activities and properties in vitro and in vivo in a ratcorneal model. See Camenisch et al., J. Biol. Chem. 277(19): 17281-17290(2002). To address the possible risk of inducing new blood vesselsfollowing in vivo administration of C-terminal ANGPTL3, in vitroangiogenic assays were examined. Briefly, primary human umbilical veinendothelial cells (HUVECs) were serum starved overnight with basalendothelial cell media. Cells were then labeled with cell tracker greenand added to pre-coated matrigel plates embedded with protein construct(indicated). Following culture for 18 hours in the presence of fulllength ANGPTL3 (50 ng/mL) or 242KQ (50 ng/mL) or bFGF (50 ng/mL) whichwas used as a positive control, the number of branch points and thetotal tube length formed was quantified using high content imaging as ameasure of angiogenic activity. In contrast to the effect seen in thepresence of full length ANGPTL3 or positive control, no significantincrease in either parameter was detected when cells were incubated with242KQ. See FIG. 2C.

CR-MSCs exist within hyaline articular cartilage and increase in numberin response to injury. Following injury to the cartilage tissue, thesecells have the capacity to participate in repair processes, but do notsufficiently lead to proper cartilage repair on their own. Patients aretherefore left with articular cartilage that lacks the proper ability tosupport painless joint movements and often require surgical interventionand/or a joint replacement to maintain their quality of life. We havefound ANGPTL3 and in particular engineered protease resistant ANGPTL3peptides have the ability to direct the differentiation of human CR-MSCsinto chondrocytes, specifically secreting hyaline articular cartilageproteins type II collagen and Sox9 while inhibiting the fibroticcartilage formation noted by expression of type X collagen.

No expression of ANGPTL3 has been reported to our knowledge nor observedin our studies using western blotting in human chondrocytes, human MSCsor human synovial fibroblasts. In rodent joints, little to no expressionwas found through immunohistochemistry (IHC). However, in humanosteoarthritic synovial fluid (n=2), low level ANGPTL3 (1.3-6.0 ng/mL)was detected by enzyme-linked immunosorbent assay (ELISA), suggesting ina compromised joint, systemically circulating protein can enter thesynovial cavity.

Example 4: In Vivo Analysis of Constructs

Mouse Acute Injury Surgical Model.

Surgical transection of the anterior cruciate ligament (ACL), medialmeniscal tibial ligament (MMTL), and medial collateral ligament (MCL) ofthe right knee from C57BL/6 mice (n=12/group) was performed to induceinstability in the knee joint and thus lead to an OA phenotype, adaptedfrom the previously described model Glasson, S. S., et al.,Osteoarthritis Cartilage 15, 1061 (2007). To evaluate a potentialtherapeutic benefit of ANGPTL3 treatment, 15 weeks following surgery,mice were dosed intra-articularly as indicated in FIG. 6A once/per weekon weeks 17-19: mANGPTL3 dose=200 ng/knee. Quantitative assessments ofthe tibial plateau were made on a 0-4 scale, 0 being normal and 5 beingsevere osteoarthritis (full thickness disruption of the cartilage). Twosections from each mouse were blindly graded by 2 independent observers(FIG. 6B).

Alleviation of osteoarthritis induced pain for animals was measured byincapacitance testing, or determining the percentage of time the mousestood on a surgically treated leg vs the non-treated leg using anincapacitance monitoring device. FIG. 7 depicts results of readouts,representing pain response on days 35 and 56 after surgery were reportedas a % weight bearing on the surgical limb versus the non surgical limb.Treatment depicts results of animals dosed as described above with fulllength murine ANGPTL3 (WT17-460) or C-terminal human ANGPTL3(WT225-460).

Mouse Chronic OA Model (Collagenase VII Induced)

Another widely used animal model of osteoarthritis, the collagenaseVII-induced chronic joint injury model, was used to evaluate in vivoefficacy of constructs. The model and evaluation was performed aspreviously described. See van der Kraan, P. M., et al., Am. J. Pathol.135, 1001 (1989); and Johnson, K., et al., Science 336, 717 (2012).Briefly, a three (3) day period of inflammation is followed bycollagenase induced destabilization of the joint, resulting in mild tomoderate cartilage destruction. Intra-articular administration ofconstructs was carried out following induction in the knee once/week forthree weeks, beginning 3 weeks after addition of collagenase VII. Forty(42) days following treatment, joints were collected and sectioned.Histological joint severity scoring of femoral and tibial plateauallowed quantification of the tissue repair. The severity of the jointscore was determined through histological scoring as described above.FIG. 8 depicts repair with 225WT, 225KQ, 228KQ, 233KQ, and 241KQconstructs. To confirm the presence of protein in the joint (long termintra-articular retention), tissue was fixed and stained for thepresence of the WT protein construct through immunohistochemistry.Analysis confirmed the presence of protein indicating intra-articularretention of ANGPTL3 (with no effects seen on lipid/triglyceride,assessed using a standard metabolic panel, data not shown.)

Histological analysis and grading on Safranin 0 stained sections of themedial tibial plateau (for detection of proteoglycan at the injury site,as described above) revealed regeneration in cartilage matrix (data notshown). Qualitative analysis confirmed replacement of proteoglycanssimilar to levels seen in a naïve mouse, while vehicle controls did notshow similar replacement. Tissue sections were also stained as describedabove for type II collagen 8 weeks following injection of the injury.Qualitative analyses confirmed an increase of type II collagen in jointstreated with construct similar to levels seen in a naïve mouse; whilevehicle treated controls did not show similar increase (data not shown).

Rat Meniscal Tear Model

A rat surgical injury model was also used to evaluate in vivo efficacyof constructs. The model and evaluation was initially performed aspreviously described Gerwin N. et al. Osteoarthritis Cartilage. Suppl 3:S24 (2010). Briefly, skin was shaven over a knee joint and the medialcollateral ligament (MCL) was isolated through an incision, and the MCLwas stabilized and a distal cut of the meniscus made using a scalpel. Onweeks 1, 2 and 3 following surgery protein construct or vehicle controlwas injected intra-articularly, then joints were collected and sectionedat 4 and 6 weeks after surgery. Histological joint severity scoring offemoral and tibial plateau were performed for quantification of thetissue repair as described above. Data is shown for the 6 weeksanalyses.

Healthy hyaline cartilage replaced damage following treatment.Histological analysis and grading of the lateral tibial plateau ofsafranin 0 stained cartilage were performed as described above andquantified Results demonstrated animals treated with 242KQ constructrevealed regeneration in cartilage matrix and replacement ofproteoglycans similar to levels seen in a naïve rat, while vehiclecontrols did not show similar replacement. See FIG. 9. Similar resultswere seen with 225WT.

A slightly altered surgically induced meniscal tear model from thatdescribed above was used to initiate cartilage damage in male Lewis ratsin order to test the efficacy of 242KQ in promoting cartilage repair invivo. Surgery on rats was performed to completely sever the medialcollateral ligament and the medial meniscus to destabilize the joint sothat future weight bearing would lead to rapid degeneration of thecartilage. An incision was made to sever the ligament on both sides ofthe needle, thus ensuring a complete cut. A scalpel blade was then usedto slip under the patellar ligament into the synovial space and thepointed tip was used to cut the meniscus. A successful cut wasaccomplished when the joint dislocated laterally. One week aftersurgery, rats were dosed by intra-articular injection of 242KQ or salinein a volume of 25 uL into the intra-articular synovial space.

Twenty eight days after meniscal tear surgery and twenty one days postintra-articular injection of saline or construct, study animals wereeuthanized and affected joints were harvested for analysis, fixed in 10%formalin in PBS, decalcified with formic acid, and embedded in paraffinprior to sectioning. Coronal sections were cut and stained for SafraninO or left unstained for future immunohistochemical staining. Analysisrevealed that the medial tibial plateau had the greatest amount ofcartilage damage and it was decided to evaluate only this area of thejoint for efficacy of 242KQ. Using the OARSI scoring system, cartilageseverity scores were assigned for six sections across the width of thetibial cartilage for each animal (N=10) in a blinded manner. Scoring wasdone twice at different time-points and the scores were then averaged tocreate a score of cartilage damage. Additionally, objective scoringanalyses were performed with a custom script generated in Matlab. Thealgorithm identified the articular cartilage surfaces and objectivelyquantified additional cartilage parameters (zonal analyses, safranin Ointensity, cartilage area, cartilage thickness). Results are depicted inFIG. 10A.

Structural repair of cartilage is not always associated with relief ofpain, at least in humans. Although rodent physiology and gait aresignificantly different than humans, 242KQ was evaluated to determine ifthere was any improvement in the gait or length of time spent on thesurgical limb after treatment. Incapacitance monitoring was performed onrats treated with 242KQ. Rats were subjected to the modified meniscalsurgery as described above. One week following the surgery, 242KQ wasinjected into the synovial space. On day 28, the rats were placed on anincapacitance monitor on their hind limbs and 30 subsequent readingswere taken over 10 minutes for each rat to determine the percent of timespent (weight distribution) on each hind limb. These data give anindication of the pain-induced weight redistribution It was determinedthat in the rat meniscal tear model, treatment with 242KQ one weekfollowing surgery led to a partial restoration of the equal weightbearing capacity of the rats. See FIG. 10B.

One of the primary challenges during spontaneous or surgical cartilagerepair is the replacement of hyaline articular cartilage with fibroticcartilage. To explore the type of cartilage repair mediated by ANGPTL3,sections from the rat knees collected from the rat meniscal tear studyperformed above were stained for the presence of type II collagen (toindicate hyaline articular cartilage) and type X collagen (to indicatefibrotic cartilage). After a single injection of 20 μg of 242KQ, therewas a qualitative reduction in the amount of type X collagen expression.

Long term retention of 242KQ following intravenous and intra-articularinjection into rat knees was determined through ¹²⁴I labeling of proteinand administration followed by PET/uCT imaging to monitor retention.See, Gerwin, N., et al. (2006) Advanced drug delivery reviews 58,226-242. The mean residence time (MRT) after IA injection of 242KQ intothe joint was determined to be ˜17.3 h which is significantly increasedover the standard 2-3 h reported (See TABLE 6)

TABLE 6 Persistence of ¹²⁴I 242KQ Dose C_(max) AUC_(0-inf) CL Vss MRTT_(1/2) Route (μg) (μg/mL) (hr*μg/mL) (mL/h) (mL) (h) (h) IV 164.2 129.322.1 7.4 53.4 7.2 12.4 IA 38.3 0.2 1.9 — — 17.3 7.2

Doe Partial Menisectomy Joint Injury Model

We also evaluated ANGPTL3 activity in a canine joint injury model. Themodel was performed and evaluations performed as described in Connor, J.R., et al., Osteoarthritis and cartilage/OARS, Osteoarthritis ResearchSociety 17, 1236-1243 (2009). Briefly, skin was shaven over a knee jointand the medial collateral ligament (MCL) was isolated through anincision, and the MCL was stabilized and a distal cut of the meniscusmade using a scalpel. Four (4) days following surgery, animals receivedeither twice weekly dosing (1.5 ug or 15 ug), or a single dose (30 ug)of the protein construct (full length canine ANGPTL3) on day 7 orvehicle control (injected intra-articularly). Dogs were euthanized onday 28 and the knees were subjected to histological, sectioning andgrading as described above for the rat and mouse experiments. FIG. 10depicts the Total gross score of the repair associated with treatment ofcanine ANGPTL3. Upon histological grading and evaluation of the dogjoint sections stained with Safranin 0, areas where the most severecartilage loss took place in the saline groups was the portion of thejoint that had the largest reduction in lesion area following a single30 μg dose of cANGPTL3.

It is understood that the examples and embodiments described herein arefor illustrative purposes and that various modifications or changes inlight thereof will be suggested to persons skilled in the art and are tobe included within the spirit and purview of this application and scopeof the appended claims.

SEQUENCES SEQ ID Construct Sequence  1 HumanMFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGH ANGPTL3GLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE  2 Humanttccagaagaaaacagttccacgttgcttgaaattgaaaatcaagataaaaatgt ANGPTL3tcacaattaagctccttctttttattgttcctctagttatttcctccagaattga REFSEQtcaagacaattcatcatttgattctctatctccagagccaaaatcaagatttgctatgttagacgatgtaaaaattttagccaatggcctccttcagttgggacatggtcttaaagactttgtccataagacgaagggccaaattaatgacatatttcaaaaactcaacatatttgatcagtctttttatgatctatcgctgcaaaccagtgaaatcaaagaagaagaaaaggaactgagaagaactacatataaactacaagtcaaaaatgaagaggtaaagaatatgtcacttgaactcaactcaaaacttgaaagcctcctagaagaaaaaattctacttcaacaaaaagtgaaatatttagaagagcaactaactaacttaattcaaaatcaacctgaaactccagaacacccagaagtaacttcacttaaaacttttgtagaaaaacaagataatagcatcaaagaccttctccagaccgtggaagaccaatataaacaattaaaccaacagcatagtcaaataaaagaaatagaaaatcagctcagaaggactagtattcaagaacccacagaaatttctctatcttccaagccaagagcaccaagaactactccctttcttcagttgaatgaaataagaaatgtaaaacatgatggcattcctgctgaatgtaccaccatttataacagaggtgaacatacaagtggcatgtatgccatcagacccagcaactctcaagtttttcatgtctactgtgatgttatatcaggtagtccatggacattaattcaacatcgaatagatggatcacaaaacttcaatgaaacgtgggagaactacaaatatggttttgggaggcttgatggagaattttggttgggcctagagaagatatactccatagtgaagcaatctaattatgttttacgaattgagttggaagactggaaagacaacaaacattatattgaatattctttttacttgggaaatcacgaaaccaactatacgctacatctagttgcgattactggcaatgtccccaatgcaatcccggaaaacaaagatttggtgttttctacttgggatcacaaagcaaaaggacacttcaactgtccagagggttattcaggaggctggtggtggcatgatgagtgtggagaaaacaacctaaatggtaaatataacaaaccaagagcaaaatctaagccagagaggagaagaggattatcttggaagtctcaaaatggaaggttatactctataaaatcaaccaaaatgttgatccatccaacagattcagaaagctttgaatgaactgaggcaaatttaaaaggcaataatttaaacattaacctcattccaagttaatgtggtctaataatctggtattaaatccttaagagaaagcttgagaaatagattttttttatcttaaagtcactgtctatttaagattaaacatacaatcacataaccttaaagaataccgtttacatttctcaatcaaaattcttataatactatttgttttaaattttgtgatgtgggaatcaattttagatggtcacaatctagattataatcaataggtgaacttattaaataacttttctaaataaaaaatttagagacttttattttaaaaggcatcatatgagctaatatcacaactttcccagtttaaaaaactagtactcttgttaaaactctaaacttgactaaatacagaggactggtaattgtacagttcttaaatgttgtagtattaatttcaaaactaaaaatcgtcagcacagagtatgtgtaaaaatctgtaatacaaatttttaaactgatgcttcattttgctacaaaataatttggagtaaatgtttgatatgatttatttatgaaacctaatgaagcagaattaaatactgtattaaaataagttcgctgtctttaaacaaatggagatgactactaagtcacattgactttaacatgaggtatcactataccttatt  3 MurineMHTIKLFLFVVPLVIASRVDPDLSSFDSAPSEPKSRFAMLDDVKILANGLLQLGH ANGPTL3GLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLRTNEIKEEEKELRRTTSTLQVKNEEVKNMSVELNSKLESLLEEKTALQHKVRALEEQLTNLILSPAGAQEHPEVTSLKSFVEQQDNSIRELLQSVEEQYKQLSQQHMQIKEIEKQLRKTGIQEPSENSLSSKSRAPRTTPPLQLNETENTEQDDLPADCSAVYNRGEHTSGVYTIKPRNSQGFNVYCDTQSGSPWTLIQHRKDGSQDFNETWENYEKGFGRLDGEFWLGLEKIYAIVQQSNYILRLELQDWKDSKHYVEYSFHLGSHETNYTLHVAEIAGNIPGALPEHTDLMFSTWNHRAKGQLYCPESYSGGWWWNDICGENNLNGKYNKPRTKSRPERRRGIYWRPQSRK LYAIKSSKMMLQPTT 4 Canine MYTIKLFLFIIPLVISSKIDRDYSSYDSVSPEPKSRFAMLDDVKILANGLLQLGHANGPTL3 GLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTNEIKEEEKELRRTTSKLQVKNEEVKNMSLELNSKVESLLEEKILLQQKVRYLEKQLTSLIKNQPEIQEHPEVTSLKTFVEQQDNSIKDLLQTVEEQYRQLNQQHSQIKEIENQLRNVIQESTENSLSSKPRAPRTTPFLHLNETKNVEHNDIPANCTTIYNRGEHTSGIYSIRPSNSQVFNVYCDVKSGSSWTLIQHRIDGSQNFNETWENYRYGFGRLDGEFWLGLEKIYSIVKQSNYILRIELEDWNDNKHYIEYFFHLGNHETNYTLHLVEITGNILNALPEHKDLVFSTWDHKAKGHVNCPESYSGGWWWHNVCGENNLNGKYNKQRAKTKPERRRGLYWKSQNGRLYSIKSTKMLIHPIDSESSE  5 EquineMYTIKLFLVIAPLVISSRIDQDYSSLDSIPPEPKSRFAMLDDVKILANGLLQLGH ANGPTL3GLKDFVHKTKGQINDIFQKLNIFDQSFYALSLQTNEIKEEEKELRRTTSKLQVKNEEVKNMSLELNSKLESLLEEKSLLQQKVKYLEEQLTKLIKNQPEIQEHPEVTSLKTFVEQQDNSIKDLLQTMEEQYRQLNQQHSQIKEIENQLRRTGIQESTENSLSSKPRAPRTTPSFHLNETKDVEHDDFPADCTTIYNRGEHTSGIYSIKPSNSQVFNVYCDVISGSSWILIQRRIDGSQNFNETWQNYKYGFGRLDFEFWLGLEKIYSIVKRSNYILRIELEDWKDNKHTIEYSFHLGNHETNYTLHLVEITGNVPNALPEHKDLVFSTWDHKAKGQLNCLESYSGGWWWHDVCGGDNPNGKYNKPRSKTKPERRRGICWKSQNGRLYTIKSTKMLIHPIDSESFELRQIKKPMN  6 BovineMYTIKLFLIIAPLVISSRTDQDYTSLDSISPEPKSRFAMLDDVKILANGLLQLGH ANGPTL3GLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTNEIKEEEKELRRATSKLQVKNEEVKNMSLELDSKLESLLEEKILLQQKVRYLEDQLTDLIKNQPQIQEYLEVTSLKTLVEQQDNSIKDLLQIVEEQYRQLNQQQSQIKEIENQLRRTGIKESTEISLSSKPRAPRTTPSFHSNETKNVEHDDIPADCTIIYNQGKHTSGIYSIRPSNSQVFNVYCDVKSGSSWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVMQSNYILRIELEDWKDKYYTEYSFHLGDHETNYTLHLAEISGNGPKAFPEHKDLMFSTWDHKAKGHFNCPESNSGGWWYHDVCGENNLNGKYNKPKAKAKPERKEGICWKSQDGRLYSIKATKMLIHPSDSENSE  7 207-455WTIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD  8 225-455WTTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTD  9228-455WT FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKST KMLIHPTD 10233-455WT EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTD 11 241-455WTGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 12 ANGPTL3KQMFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 13 ANGPTL3KSMFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 14 207KQIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 15 207KSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 16 225KQTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTDSESFE17 225KS TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTDSESFE18 225ST TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKTKPERRRGLSWKSQNGRLYSI KSTKMLIHPTDSESFE19 226KQ TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIK STKMLIHPTDSESFE20 226KS TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIK STKMLIHPTDSESFE21 228KQ FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKST KMLIHPTDSESFE 22228KS FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKST KMLIHPTDSESFE 232285T FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKTKPERRRGLSWKSQNGRLYSIKST KMLIHPTDSESFE 24233KQ EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTDSESFE 25233KS EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTDSESFE 26241KQ GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 27 241KSGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 28 242KQIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 29 242KSIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 30 225-455KQTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTD 31225-455KS TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTD 32226-455KQ TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIK STKMLIHPTD 33226-455KS TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIK STKMLIHPTD 34228-455KQ FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKST KMLIHPTD 35228-455KS FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKST KMLIHPTD 36233-455KQ EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTD 37 233-455KSEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTD 38 241-455KQGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 39 241-455KSGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 40 242-455KQIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 41 242-455KSIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 42 CanineFLHLNETKNVEHNDIPANCTTIYNRGEHTSGIYSIRPSNSQVFNVYCDVKSGSSW 227KQTLIQHRIDGSQNFNETWENYRYGFGRLDGEFWLGLEKIYSIVKQSNYILRIELEDWNDNKHYIEYFFHLGNHETNYTLHLVEITGNILNALPEHKDLVFSTWDHKAKGHVNCPESYSGGWWWHNVCGENNLNGKYNKQRAQTKPERRRGLYWKSQNGRLYSIKST KMLIHPIDSESSE 43Canine FLHLNETKNVEHNDIPANCTTIYNRGEHTSGIYSIRPSNSQVFNVYCDVKSGSSW 227KSTLIQHRIDGSQNFNETWENYRYGFGRLDGEFWLGLEKIYSIVKQSNYILRIELEDWNDNKHYIEYFFHLGNHETNYTLHLVEITGNILNALPEHKDLVFSTWDHKAKGHVNCPESYSGGWWWHNVCGENNLNGKYNKQRASTKPERRRGLYWKSQNGRLYSIKST KMLIHPIDSESSE 44Nucleic ACTACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTC acidCTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGC sequenceCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGT 225WTAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAAATCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 45 NucleicACTACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTC acidCTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGC sequenceCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGT 225KQAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCACAATCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 46 NucleicACTACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTC acidCTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGC sequenceCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGT 225KSAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAGCTCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 47 NucleicACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTG acidCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGCCAT sequenceCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGTAGT 226KQCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCACAATCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 48 NucleicACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTG acidCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGCCAT sequenceCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGTAGT 226KSCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAGCTCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 49 NucleicTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGAAT acidGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGCCATCAGACC sequenceCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGTAGTCCATGG 228KQACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCACAATCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 50 NucleicTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGAAT acidGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGCCATCAGACC sequenceCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGTAGTCCATGG 228KSACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAGCTCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 51 NucleicGAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGAATGTACCACCATTTATA acidACAGAGGTGAACATACAAGTGGCATGTATGCCATCAGACCCAGCAACTCTCAAGT sequenceTTTTCATGTCTACTGTGATGTTATATCAGGTAGTCCATGGACATTAATTCAACAT 233KQCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCACAATCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 52 NucleicGAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGAATGTACCACCATTTATA acidACAGAGGTGAACATACAAGTGGCATGTATGCCATCAGACCCAGCAACTCTCAAGT sequenceTTTTCATGTCTACTGTGATGTTATATCAGGTAGTCCATGGACATTAATTCAACAT 233KSCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAGCTCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 53 NucleicGGCATTCCTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCA acidTGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTAT sequenceATCAGGTAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTC 241KQAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCACAATCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 54 NucleicGGCATTCCTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCA acidTGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTAT sequenceATCAGGTAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTC 241KSAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAGCTCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 55 NucleicATTCCTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGT acidATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATC sequenceAGGTAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAAT 242KQGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCACAATCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 56 NucleicATTCCTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGT acidATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATC sequenceAGGTAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAAT 242KSGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAGCTCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 57 NucleicTTTTTGCATCTCAACGAAACGAAGAATGTCGAACACAACGACATTCCGGCAAATT acidGCACAACTATCTACAATAGAGGCGAACATACGTCCGGTATCTACTCCATTAGACC sequenceTTCAAACAGCCAGGTATTCAATGTGTACTGCGATGTAAAGTCAGGATCGTCATGG c227KQACACTGATCCAGCATAGGATCGACGGGTCCCAGAACTTCAACGAGACATGGGAGAACTACCGCTATGGATTTGGAAGGCTGGATGGGGAGTTCTGGTTGGGACTTGAGAAAATCTACAGCATTGTGAAGCAGTCGAACTACATTCTCCGGATTGAACTGGAGGACTGGAATGACAACAAACACTACATCGAGTATTTCTTTCATCTCGGCAACCATGAAACGAATTACACCTTGCACCTTGTGGAAATCACGGGCAACATTTTGAACGCGCTGCCAGAACACAAAGACCTGGTGTTTTCGACATGGGATCACAAAGCAAAGGGGCACGTGAACTGTCCCGAATCATATAGCGGGGGATGGTGGTGGCACAATGTCTGTGGTGAGAACAATCTCAACGGGAAATACAATAAGCAGCGAGCTCAGACGAAACCCGAGCGGCGGAGAGGTCTGTATTGGAAGTCGCAGAATGGACGCCTGTATTCGATCAAATCGACGAAAATGCTCATCCACCCCATCGACTCCGAATCGTCGGAG 58 207KdelIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 59 225KdelTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIK STKMLIHPTDSESFE60 226Kdel TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKS TKMLIHPTDSESFE61 228Kdel FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTK MLIHPTDSESFE 62233Kdel EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHP TDSESFE 63241Kdel GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 64 242KdelIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 65 225-TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG 455KdelSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIK STKMLIHPTD 66226- TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS 455KdelPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKS TKMLIHPTD 67228- FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW 455KdelTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTK MLIHPTD 68 233-EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH 455KdelRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHP TD 69 241-GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF 455KdelNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 70 242-IPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN 455KdelETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 71 hANGPTL1MKTFTWTLGVLFFLLVDTGHCRGGQFKIKKINQRRYPRATDGKEEAKKCAYTFLVP 1-491EQRITGPICVNTKGQDASTIKDMITRMDLENLKDVLSRQKREIDVLQLVVDVDGNIVNEVKLLRKESRNMNSRVTQLYMQLLHEIIRKRDNSLELSQLENKILNVTTEMLKMATRYRELEVKYASLTDLVNNQSVMITLLEEQCLRIFSRQDTHVSPPLVQVVPQHIPNSQQYTPGLLGGNEIQRDPGYPRDLMPPPDLATSPTKSPFKIPPVTFINEGPFKDCQQAKEAGHSVSGIYMIKPENSNGPMQLWCENSLDPGGWTVIQKRTDGSVNFFRNWENYKKGFGNIDGEYWLGLENIYMLSNQDNYKLLIELEDWSDKKVYAEYSSFRLEPESEFYRLRLGTYQGNAGDSMMWHNGKQFTTLDRDKDMYAGNCAHFHKGGWWYNACAHSNLNGVWYRGGHYRSKHQDGIFWAEYRGGSYSLRAVQMMIKPID 72 CTFINEGPFKDCQQAKEAGHSVSGIYMIKPENSNGPMQLWCENSLDPGGWTVIQKRTD hANGPTL1GSVNFFRNWENYKKGFGNIDGEYWLGLENIYMLSNQDNYKLLIELEDWSDKKVYAE 271-491YSSFRLEPESEFYRLRLGTYQGNAGDSMMWHNGKQFTTLDRDKDMYAGNCAHFHKGGWWYNACAHSNLNGVWYRGGHYRSKHQDGIFWAEYRGGSYSLRAVQMMIKPID 73 hANGPTL4MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQLGQGLR 1-406EHAERTRSQLSALERRLSACGSACQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQA TTMLIQPMAAEAAS74 CT SRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGS hANGPTL4VDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFS 179-406VHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAA EAAS

What is claimed is:
 1. An isolated polynucleotide encoding a polypeptidecomprising an amino acid sequence that has at least 95% amino acidsequence identity to an amino acid sequence selected from any one of SEQID NOs: 58-70, wherein the amino acid at position 423, as determinedwith reference to SEQ ID NO:1, is deleted, and wherein the polypeptidehas chondrogenic activity.
 2. The isolated polynucleotide according toclaim 1, which is complementary DNA (cDNA) or messenger RNA (mRNA). 3.The isolated polynucleotide according to claim 1, wherein the encodedpolypeptide further comprises a secretion signal sequence.
 4. Theisolated polynucleotide according to claim 1, wherein the encodedpolypeptide further comprises a fusion domain selected from the groupconsisting of a polyhistidine, Glu-Glu, glutathione S transferase (GST),thioredoxin, protein A, protein G, an immunoglobulin heavy chainconstant region (Fc), maltose binding protein (MBP), a human serumalbumin (HSA), a FLAG tag, influenza virus hemagglutinin (HA), and ac-myc tag.
 5. The isolated polynucleotide according to claim 1, which iscodon-optimized for expression in a host cell.
 6. The isolatedpolynucleotide according to claim 1, which is operably linked to apromoter or other regulatory sequence.
 7. A cloning or expression vectorcomprising one or more polynucleotides according to claim 1, wherein thevector is suitable for the recombinant production of the encodedpolypeptide.
 8. An isolated host cell, comprising the cloning orexpression vector according to claim
 7. 9. The isolated host cell ofclaim 8, which is a mammalian host cell, a bacterial host cell, a yeasthost cell, or an insect host cell.
 10. A process for the production of apolypeptide comprising an amino acid sequence that has at least 95%amino acid sequence identity to an amino acid sequence selected from anyone of SEQ ID NOs: 58-70, wherein the amino acid at position 423, asdetermined with reference to SEQ ID NO: 1, is deleted, and wherein thepolypeptide has chondrogenic activity, said process comprising culturingthe host cell according to claim 9 under conditions sufficient toexpress the polypeptide, and thereafter purifying and recovering thepolypeptide from the host cell culture.
 11. A kit comprising thepolynucleotide according to claim 1, wherein the kit comprises: a firstcontainer comprising the polynucleotide; and a second container havingan aqueous reconstitution formula.
 12. The kit according to claim 11,wherein one of the containers comprises a single chambered pre-filledsyringe.
 13. The kit according to claim 11, wherein the containers areencompassed as a multi-chambered pre-filled syringe.